Agent that modulates physiological condition of pests, involved in insect choline acetyltransferase activity

ABSTRACT

The present invention provides an agent that modulates physiological condition of pests, wherein the agent has an ability to modulate the activity of an insect choline acetyltransferase; a method for assaying pesticidal activity of a test substance, which comprises measuring the activity of a choline acetyltransferase in a reaction system in which the choline acetyltransferase contacts with a test substance, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/743,866 filed Oct. 8, 2010, now pending, which is a 371 ofPCT/JP2008/071748, filed Nov. 21, 2008, which claims priority toJapanese application 2007-302699, filed Nov. 22, 2007. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 19, 2010, is named Q118912.txtand is 13,556 bytes in size.

TECHNICAL FIELD

The present invention relates to an agent that modulates physiologicalcondition of pests, involved in insect choline acetyltransferaseactivity, and the like.

BACKGROUND ART

Choline acetyltransferase (acetyl CoA:choline O-acetyltransferase, EC2.3.1.6; synonym: Choline acetylase, Choline O-acetyltransferase; ChAT)is the enzyme responsible for catalyzing the biosynthesis of theneurotransmitter acetylcholine from its precursors, acetyl-coenzyme A(acetyl-CoA) and choline. Acetylcholine was the first neurotransmitterto be reported and plays a pivotal role in such fundamental brainprocesses as learning, memory and sleep. Acetylcholine functions in thecholinergic neurons of the peripheral and central nervous systems. Inthe peripheral nervous system acetylcholine stimulates musclecontraction through the neuron-muscular junction and in the centralnervous system acetylcholine facilitates learning and short-term memoryformation.

There have been reports on choline acetyltransferase from invertebratesincluding insects as follows. The C. elegans choline acetyl transferaseis enriched in synaptic regions of cholinergic neurons (Duerr et al.,Midwest Worm Meeting abstract 39). A loss-of-function mutation in C.elegans choline acetyltransferase gene leads to growth arrest at L1larval stages and death (Yook and Jorgensen, West Coast Worm Meetingabstract 260). A severe reduction-of-function mutation in C. eleganscholine acetyltransferase gene leads to slow growth, small body size andan irregular defecation cycle (Rand and Russell, Genetics,106(2):227-248, 1984). The mutation leads to resistance to acetylcholineesterase inhibitors such as aldicarb or trichlorfon presumably due toundersynthesis of acetylcholine (Rand and Russell, Genetics,106(2):227-248, 1984).

Choline acetyltransferase is widely distributed in the central nervoussystem of Drosophila melanogaster throughout all developmental stages(Gorczyca and Hall, J. Neurosci., 7(5): 1361-1369, 1987). Recessivenon-conditional loss-of-function mutants for the Drosophila melanogastercholine acetyltransferase gene die at the late embryonic stage(Greenspan, J. Comp. Physiol., 137(1):83-92, 1980). Temperaturesensitive reduction-of-function mutants for Drosophila melanogastercholine acetyltransferase become paralysed after incubation at arestrictive temperature (Kitamoto et al., J. Neurobiol., 42(2):161-171,2000). Using temperature-sensitive loss-of-function mutation at the D.melanogaster choline acetyltransferase locus, it has been shown thatnormal acethylcholine metabolism is not required for the initialformation of the nervous system but is required for the subsequentmaintenance of its structural integrity and function (Chase and Kankel,Dev. Biol., 125(2):361-380, 1988).

Discovery of agricultural chemicals has traditionally been based on arandom screening process, often directly testing the effects of specificchemicals on whole organisms, such as insects, fungi and/or plants, anddetermining biological activity. Once chemical compounds with theappropriate biological activity are discovered, more intense research isrequired to specifically determine the mode of action or site of actionof these compounds at the molecular level, in order to predict safetyand environmental load of these compounds.

DISCLOSURE OF INVENTION

This invention describes a more target-based approach of screeningagricultural chemicals, whereby compounds are screened against aspecific target that has been identified as biologically and/orphysiologically relevant with intent of chemically interfering with thetarget site to control pest organisms.

Specifically, this invention describes that an agent that modulatesphysiological condition of pests and having an ability to modulate theactivity of an insect choline acetyltransferase is useful to controlpests.

That is, the present invention provides:

1. An agent that modulates physiological condition of pests, whereinsaid agent has an ability to modulate the activity of an insect cholineacetyltransferase;

2. An agent according to item 1, wherein said choline acetyltransferaseis a cotton aphid choline acetyltransferase;

3. An agent according to item 1, wherein said agent is a pesticidalagent;

4. An agent according to item 1, wherein said ability to modulate theactivity of an insect choline acetyltransferase is an ability to inhibita reaction of the insect choline acetyltransferase with acetyl-CoA andcholine;

5. A pesticidal agent which comprises a substance that has an ability tomodulate the activity of an insect choline acetyltransferase or anagriculturally acceptable salt of the substance as an active ingredient;

6. A pesticidal agent according to item 5, wherein said substance has anability to inhibit a reaction of the insect choline acetyltransferasewith acetyl-CoA and choline;

7. A pesticidal agent according to item 6, wherein said substance has anability to inhibit the reaction of the insect choline acetyltransferasewith acetyl-CoA and choline in a cell-free system, wherein in thepresence of said substance of 10 micro M or more the activity of saidcholine acetyltransferase is lower than that in the absence of saidsubstance;

8. A pesticidal agent according to item 6, wherein said substance has anability to inhibit a reaction of the insect choline acetyltransferasewith acetyl-CoA and choline in a cell-free system with an IC₅₀ of 100micro M or less;

9. A method for assaying pesticidal activity of a test substance, whichcomprises:

(1) a first step of measuring the activity of a cholineacetyltransferase selected from the following group A in a reactionsystem in which said choline acetyltransferase contacts with a testsubstance; and

(2) a second step of evaluating the pesticidal activity of said testsubstance based on the difference obtained by comparing the activitymeasured in the first step with the activity of a control:

<Group A>

(a) a protein comprising the amino acid sequence of SEQ ID NO: 1;

(b) a protein comprising an amino acid sequence with deletion, additionor substitution of one or more amino acids in the amino acid sequence ofSEQ ID NO: 1, wherein said protein has choline acetyltransferaseactivity;

(c) a protein comprising an amino acid sequence that has sequenceidentity of 50% or more to the amino acid sequence of SEQ ID NO: 1,wherein said protein has choline acetyltransferase activity;

(d) a protein comprising an amino acid sequence that has sequencesimilarity of 75% or more to the amino acid sequence of SEQ ID NO: 1,wherein said protein has choline acetyltransferase activity;

(e) a protein comprising the amino acid sequence encoded by thenucleotide sequence of SEQ ID NO: 2 or 3;

(f) a protein comprising an amino acid sequence encoded by a nucleotidesequence that has sequence identity of 50% or more to the nucleotidesequence of SEQ ID NO: 2 or 3, wherein said protein has cholineacetyltransferase activity;

(g) a protein comprising an amino acid sequence encoded by apolynucleotide, wherein said polynucleotide hybridizes under a stringentcondition to a polynucleotide comprising a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 2 or 3, andwherein said protein has choline acetyltransferase activity;

(h) a protein comprising an amino acid sequence of an insect cholineacetyltransferase; and

(i) a protein comprising an amino acid sequence of a cotton aphidcholine acetyltransferase;

10. A method for screening a pesticidal substance, which comprisesselecting a substance having the pesticidal activity that is evaluatedby the method of item 9;

11. A pesticidal agent which comprises a substance selected by themethod of item 10 or agriculturally acceptable salts thereof as anactive ingredient;

12. A method for controlling pests which comprises applying an effectiveamount of the pesticidal agent of item 5,6,7,8 or 11 to the pest,habitat of the pest or plant to be protected from the pest;

13. A method for controlling pests which comprises: identifying asubstance having the pesticidal activity that is evaluated by the methodof item 9, and

contacting the pest with the identified pesticidal substance;

14. An insect choline acetyltransferase comprising an amino acidsequence selected from the following group B:

<Group B>

(a) the amino acid sequence of SEQ ID NO: 1;

(b) an amino acid sequence with deletion, addition or substitution ofone or more amino acids in the amino acid sequence of SEQ ID NO: 1,wherein said amino acid sequence has choline acetyltransferase activity;

(c) an amino acid sequence that has sequence identity of 50% or more tothe amino acid sequence of SEQ ID NO: 1, wherein said amino acidsequence has choline acetyltransferase activity;

(d) an amino acid sequence that has sequence similarity of 75% or moreto the amino acid sequence of SEQ ID NO: 1, wherein said amino acidsequence has choline acetyltransferase activity;

(e) the amino acid sequence encoded by the nucleotide sequence of SEQ IDNO: 2 or 3;

(f) an amino acid sequence encoded by a nucleotide sequence that hassequence identity of 50% or more to the nucleotide sequence of SEQ IDNO: 2 or 3, wherein said amino acid sequence has cholineacetyltransferase activity;

(g) an amino acid sequence encoded by a polynucleotide, wherein saidpolynucleotide hybridizes under a stringent condition to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 2 or 3, wherein said amino acidsequence has choline acetyltransferase activity; and

(h) an amino acid sequence of a cotton aphid choline acetyltransferase;

15. Use of an insect choline acetyltransferase as a reagent thatprovides an indicator to evaluate pesticidal activity;

16. Use of an insect choline acetyltransferase of item 14 as a reagentthat provides an indicator to evaluate pesticidal activity;

17. A polynucleotide which comprises a nucleotide sequence encoding anamino acid sequence of a choline acetyltransferase of item 14;

18. A polynucleotide according to item 17, which comprises thenucleotide sequence of SEQ ID NO: 2 or 3;

19. A polynucleotide which comprises a nucleotide sequence complementaryto a nucleotide sequence of a polynucleotide of item 17 or 18;

20. A polynucleotide which comprises:

a partial nucleotide sequence of a polynucleotide of item 17 or 18; or

a nucleotide sequence complementary to said partial nucleotide sequence;

21. A polynucleotide according to item 20, which comprises thenucleotide sequence of SEQ ID NO: 4 or 5;

22. A method for obtaining a polynucleotide comprising a nucleotidesequence encoding an amino acid sequence of a choline acetyltransferase,which comprises:

amplifying a desired polynucleotide by polymerase chain reaction usingas a primer a polynucleotide of item 20 or 21;

identifying the desired polynucleotide amplified; and

recovering the identified polynucleotide;

23. A method for obtaining a polynucleotide comprising a nucleotidesequence encoding an amino acid sequence of a choline acetyltransferase,which comprises:

detecting a desired polynucleotide by hybridization using as a probe apolynucleotide of item 19, 20 or 21;

identifying the desired polynucleotide detected; and

recovering the identified polynucleotide;

-   24. A circular polynucleotide comprising a nucleotide sequence of a    polynucleotide of item 17 or 18, wherein said nucleotide sequence is    operably linked to a bacteriophage promoter;

25. A circular polynucleotide according to item 24, wherein saidpromoter is a T7 RNA polymerase gene promoter;

26. A circular polynucleotide according to item 24 or 25, wherein saidpolynucleotide comprises a replication origin for autonomous replicationin a host cell;

27. A method for producing a circular polynucleotide, which comprisesligating a polynucleotide of item 17 or 18 into a vector;

28. A transformant in which a polynucleotide of item 17 or 18 isintroduced;

29. A transformant according to item 28, wherein said transformant is atransformed E. coli;

30. A method for producing a transformant, which comprises introducing apolynucleotide of item 17 or 18 into a host cell;

31. A method for producing a choline acetyltransferase, which comprisesa step of culturing the transformant of item 28 or 29 and recovering aproduced choline acetyltransferase;

32. Use of a choline acetyltransferase of item 14 or a polynucleotide ofany one of items 17 to 21 as a research tool;

33. Use according to item 32, wherein the research tool is anexperimental tool for screening a pesticidal substance; and

34. A system which comprises:

a means to input, store and manage a data information of an ability oftest substances, wherein said ability is an ability to modulate theactivity of an insect choline acetyltransferase;

a means to query and retrieve the data information based on a desiredcriterion; and

a means to display and output the result which is queried and retrieved.

MODES FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below.

In the present invention, the “pests” indicates small animals whichcause harm or discomfort to life of the people by harming man andanimals directly or by damaging crops. Examples thereof includearthropod such as insects, mites and ticks and Nematoda, and typicalexamples of which are as follows:

Hemiptera:

Delphacidae such as Laodelphax striatellus, Nilaparvata lugens andSogatella furcifera, Deltocephalidae such as Nephotettix cincticeps andEmpoasca onukii, Aphididae such as Aphis gossypii and Myzus persicae,Pentatomidae, Aleyrodidae such as Trialeurodes vaporariorum, Bemisiatabaci and Bemisia argentifolli, Coccidae, Tingidae, Psyllidae, etc.

Lepidoptera:

Pyralidae such as Chilo suppressalis, Cnaphalocrocis medinalis, Ostrinianubilalis and Parapediasia teterrella, Noctuidae such as Spodopteralitura, Spodoptera exigua, Pseudaletia separata, Mamestra brassicae,Agrotis ipsilon, Trichoplusia spp., Heliothis spp., Helicoverpa spp. andEarias spp., Pieridae such as Pieris rapae crucivora, Tortricidae suchas Adoxophyes orana fasciata, Grapholita molesta and Cydia pomonella,Carposinidae such as Carposina niponensis, Bucculatricidae such asLyonetia clerkella, Gracillariidae such as Phyllonorycter ringoniella,Phyllocnistidae such as Phyllocnistis citrella, Yponomeutidae such asPlutella xylostella, Gelechiidae such as Pectinophora gossypiella,Arctiidae, Tineidae, etc.

Diptera:

Culex such as Culex pipiens pallens, Cules tritaeniorhynchus and Culexquinquefasciatus, Aedes such as Aedes aegypti and Aedes albopictus,Anopheles such as Anophelinae sinensis, Chironomidae, Muscidae such asMusca domestica and Muscina stabulans, Calliphoridae, Sarcophagidae,Fannia canicularis, Anthomyiidae such as Delia Platura and Deliaantigua, Trypetidae, Drosophilidae, Psychodidae, Simuliidae, Tabanidae,Stomoxyidae, Agromyzidae, etc.

Coleoptera:

Diabrotica such as Diabrotica virgifera virgifera and Diabroticaundecimpunctata howardi, Scarabaeidae such as Anomala cuprea and Anomalarufocuprea, Curculionidae such as Sitophilus zeamais, Lissorphoptrusoryzophilus and Calosobruchys chinensis, Tenebrionidae such as Tenebriomolitor and Tribolium castaneum, Chrysomelidae such as Oulema oryzae,Aulacophora femoralis, Phyllotreta striolata and Leptinotarsadecemlineata, Anobiidae, Epilachna spp. such as Epilachnavigintioctopunctata, Lyctidae, Bostrychidae, Cerambycidae, Paederusfuiscipes, etc.

Thysanoptera:

Thripidae such as Thrips spp. including Thrips palmi, Frankliniella spp.including Frankliniella occidentalis and Sciltothrips spp. includingSciltothrips dorsalis, Phlaeothripidae, etc.

Hymenoptera: Tenthredimidae, Formicidae, Vespidae, etc. Dictyoptera:Blattidae, Blattellidae, etc. Orthoptera: Acrididae, Gryllotalpidae etc.Siphonaptera:

Pulex irritans, etc.

Anoplura:

Pediculus humanus capitis, etc.

Isoptera: Termitidae, etc. Acarina:

Tetranychidae such as Tetranychus urticae, Tetranychus kanzawai,Panonychus citri, Panonychus ulmi, and Oligonychus spp., Eriophyidaesuch as Aculops pelekassi and Aculus schlechtendali, Tarsonemidae suchas Polyphagotarsonemus latus, Tenuipalpidae, Tuckerellidae, Ixodidaesuch as Haemaphysalis longicornis, Haemaphysalis flava, Dermacentortaiwanicus, Ixodes ovatus, Ixodes persulcatus and Boophilus microplus,Acaridae such as Tyrophagus putrescentiae, Dermanyssidae, Cheyletidaesuch as Dermatophagoides farinae and Dermatophagoides ptrenyssnus, suchas Cheyletus eruditus, Cheyletus malaccensis and Cheyletus moorei,Dermanyssus spp., etc.

Nematodes:

Pratylenchus coffeae, Pratylenchus fallax, Heterodera glycines,Globodera rostochiensis, Meloidogyne hapla, Meloidogyne incognita, etc.

In the present invention, the “modulate physiological condition ofpests” indicates changing condition such as various phenomena in aliving body which are maintained for living in pests, for example,function such as aspiration, digestion, secretion, body liquidcirculation, metabolism and neurotransmission, or mechanism thereof intocondition apart from usual condition. Examples include changingcondition by cessation of aspiration so that oxygen necessary forinternal metabolism of pests will not be supplied, and changingcondition by cessation of function of neurotransmission of pests so thatvarious movements of pests will be ceased.

In the present invention, the “agent which modulates physiologicalcondition of pests” is an agent which can modulate physiologicalcondition of pests when being applied to pests.

In the present invention, the “insect choline acetyltransferase”indicates a choline acetyltransferase that occurs in insect, amongcholine acetyltransferase present in various organisms. Herein, insectis an animal classified under Animalia, Arthropoda, Insecta, andexamples of which include arthropod of the order Protura, Collembola,Diplura, Thysanura, Ephemeroptera, Odonata, Plecoptera, Grylloblattodea,Orthoptera, Phasmatodea, Dermaptera, Mantodea, Blattaria, Isoptera,Embioptera, Psocoptera, Mallophaga, Anoplura, Thysanoptera, Hemiptera,Neuroptera, Mecoptera, Trichoptera, Lepidoptera, Coleoptera, Diptera,Hymenoptera, Siphonaptera, Strepsiptera, and the like.

Choline acetyltransferase (acetyl CoA:choline O-acetyltransferase, EC2.3.1.6; synonym: Choline acetylase, Choline O-acetyltransferase; ChAT)catalyzes reaction to synthesize the neurotransmitter acetylcholine fromits precursors, acetyl-coenzyme A (acetyl-CoA) and choline.

The activity of choline acetyltransferase can be monitored using aradiometric based in vitro assay. For example, as reported by Heo Ho-Jinet al., Biosci. Biotechnol. Biochem., 67(6), 1284-1291, 2003, aftercholine acetyltransferase-catalyzed reaction on substrates of acetyl-CoAand choline using radiolabelled ¹⁴C-acetyl-CoA, the formed¹⁴C-acetylcholine is extracted with tetraphenylboron (TPB). The 2 phasesare separated and the radioactivity of the upper phase is measured in aliquid scintillation counter.

Another assay that can be used to measure the choline acetyltransferaseactivity is an absorbance assay. The principle of the absorbance assayis that choline acetyltransferase activity can be determined bymeasuring the free Coenzyme A (CoA) formed by choline acetyltransferasereaction using 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) reagent. DTNBreacts with free thiol groups in solution to produce5-thio-2-nitrobenzoic acid (TNB). TNB is yellow and has absorptionmaximum at 412 nm. This colored TNB can then be measured by absorbancyat 405 nm.

Of the assays of choline acetyltransferase activity described above, theassay using DTNB is preferable for measuring choline acetyltransferaseactivity in a large number of samples mechanically and efficiently.Specifically, after choline acetyltransferase reaction on substrates ofacetyl-CoA and choline, DTNB is added and then the free CoA formed bythe enzyme reaction is quantified by measuring absorbancy at 405 nm.

The activity of insect choline acetyltransferase can be measured bymethods similar to that described above.

Several amino acid sequences of choline acetyltransferase have beenidentified in different insect species such as D. melanogaster (isoformA, accession No. NP_(—)477004; and isoform B, accession No.NP_(—)996239), Tribolium castaneum (accession No. XP_(—)975503), Apismellifera (accession No. XP_(—)392463), Aedes aegypti (accession No.XP_(—)001660851) and Anopheles gambiae (accession No. XP_(—)312586),which can be found in public databases.

Also, several nucleotide sequences of choline acetyltransferase geneshave been identified in different insect species, such as D.melanogaster (isoform A, accession No. NM_(—)057656; isoform B,accession No. NM_(—)206517), Tribolium castaneum (accession No.XM_(—)970410), Apis mellifera (accession No. XM_(—)392463), Aedesaegypti (accession No. XM_(—)001660801) and Anopheles gambiae (accessionNo. XM_(—)312586), which can be found in public databases.

In addition, according to the methods described below, an amino acidsequence of choline acetyltransferase and a nucleotide sequence ofcholine acetyltransferase gene can be identified from a cotton aphid.The identified amino acid sequence of cotton aphid cholineacetyltransferase is shown in SEQ ID NO: 1, and the nucleotide sequenceof cotton aphid choline acetyltransferase gene is shown in SEQ ID NO: 2.

Several amino acid sequences of choline acetyltransferase have beenidentified in animals other than insect, such as Ceanorhabditis elegans(accession No. AAB88370) and Homo sapiens (accession No. NP065574),which can be found in public databases. Also, several nucleotidesequences of choline acetyltransferase genes have been identified inanimals other than insect, such as Ceanorhabditis elegans (accession No.ZC416.8) and Homo sapiens (accession No. NM_(—)020549), which can befound in public databases.

Table 1 shows Sequence identity of the amino acid sequence of cottonaphid choline acetyltransferase (SEQ ID NO: 1) and the nucleotidesequence of cotton aphid choline acetyltransferase gene (SEQ ID NO: 2)with the sequences of choline acetyltransferase and gene thereof foundin other animals.

TABLE 1 Identity of Identity of amino acid nucleotide sequence (%) vssequence (%) vs Origin of sequence SEQ ID NO: 1 SEQ ID NO: 2 Triboliumcastaneum 48.8 70.6 Aedes aegypti 44.7 69.9 Anopheles gambiae 44.5 71.4Apis mellifera 43.5 70.0 Drosophila melanogaster 43.2 70.1Strongylocentrotus purpuratus 40.0 69.5 Gallus gallus 39.8 69.1 Homosapiens 38.7 69.4 Canis familiaris 38.6 68.9 Danio renio 37.4 67.2Xenopus tropicalis 37.1 67.0 Bos taurus 36.7 66.2

An ability to modulate the activity of an insect cholineacetyltransferase refers to an ability to increase or decrease activityof an insect choline acetyltransferase, that is, means an ability toactivate a choline acetyltransferase, or an ability to inhibit activityof a choline acetyltransferase. And, a test substance may be added tothe reaction system for measuring choline acetyltransferase activity toinvestigate influence of the test substance on the cholineacetyltransferase activity.

Several substances having an ability to inhibit activity of a cholineacetyltransferase, such as Bromoacetylcholine (Sastry and Janson, J.Ocular Pharmacol., 10:203-215, 1994), Theaflavin (Sugatani et al., Int.Arch. Allergy Immunol., 134:17-28, 2004) and α-NETA (Sastry et al., J.Pharmacol. Exp. Ther., 245:72-80, 1988) have been known.

An IC₅₀ value of a test substance in the reaction means a concentrationof a test substance at which 50% of the activity of the reaction with notest substance is inhibited. The IC₅₀ value of a test substance can bedetermined by adding test substances of different concentrations to thecholine acetyltransferase activity measuring reaction system, measuringthe choline acetyltransferase activity (response) at each concentrationof added test substance (dose), producing a dose-response curve, andcalculating a concentration of the added test substance, at which thecholine acetyltransferase activity is 50% inhibited. More specifically,a dose-response curve may be produced using 4 Parameter Logistic Modelor Sigmoidal Dose-Response Model:

${f(x)} = \left( {{A + {\left( {\left( {B - A} \right)/\left( {1 + \left( {\left( {C/x} \right)\hat{}D} \right)} \right)} \right){f(x)}}} = {A + \frac{B - A}{1 + \left( {C/x} \right)^{D}}}} \right.$

to calculate the IC₅₀. Practically, the IC₅₀ value may be calculatedusing XLfit (manufactured by IDBS) which is a commercially availablecalculating software.

An agent that has an ability to modulate the activity of an insectcholine acetyltransferase is an agent containing as an active ingredienta substance having an ability to modulate the activity of an insectcholine acetyltransferase.

In the present invention, the “agent that modulates physiologicalcondition of pests, wherein the agent has an ability to modulate theactivity of an insect choline acetyltransferase” is an agent having anability to modulate the activity of insect choline acetyltransferaseidentified by the aforementioned measuring method, and means an agentthat can modulate physiological condition of pests. Preferable examplesof the agent include an agent in which an insect cholineacetyltransferase is a cotton aphid choline acetyltransferase. Inaddition, preferable examples of the agent include an agent in which anagent that modulates physiological condition of pests is a pesticidalagent. In addition, preferable examples of the agent include an agent inwhich an ability to modulate the activity of an insect cholineacetyltransferase is an ability to inhibit a reaction using acetyl-CoAand choline as substrates.

In the present invention, the “pesticidal agent” indicates an agenthaving an ability to control the pests.

Examples of a method for measuring an ability to control pests include,in addition to the methods disclosed in the present invention, a methodof measuring pesticidal activity on the pests. Specifically, forexample, the pesticidal activity can be measured according to thefollowing method.

According to the method described in Handbook of Insect Rearing Vol. 1(Elsevier Science Publishers 1985), pp. 35 to pp. 36 except that asterilized artificial feed having the composition shown in Table 2 isprepared, and a solution of a test agent in DMSO is added at 0.5% byvolume of the artificial feed and is mixed, a cotton aphid is reared,the number of surviving cotton aphids is investigated after 6 days, anda controlling value is obtained according to the following equation.

TABLE 2 Amino acid (mg/100 ml) L-Alanine 100.0 L-arginine 275.0L-Asparagine 550.0 L-Aspartic acid 140.0 L-cysteine 40.0 (hydrochloride)L-glutamic acid 140.0 L-glutamine 150.0 L-glycine 80.0 L-histidine 80.0L-isoleucine 80.0 L-leucine 80.0 L-lysine 120.0 (hydrochloride)L-methionine 80.0 L-phenylalanine 40.0 L-proline 80.0 L-serine 80.0L-threonine 140.0 L-tryptophan 80.0 L-tyrosine 40.0 L-valine 80.0Vitamins (mg/100 ml) Ascorbic acid 100.0 Biotin 0.1 Calcium 5.0pantothenate Choline 50.0 chloride Inositol 50.0 Nicotinic acid 10.0Thiamine 2.5 Others (mg/100 ml) Sucrose 12500.0 Dipotassium 1500.0hydrogen phosphate Magnesium 123.0 sulfate Cupric chloride 0.2 Ferricchloride 11.0 Manganese 0.4 chloride Zinc sulfate 0.8 (anhydrous)Adjusted to pH 6.8

Controlling value(%)={1−(Cb×Tai)/(Cai×Tb)}×100

Letters in the equation represent the following meanings.

Cb: Number of surviving worms before treatment in non-treating section

Cai: Number of surviving worms at observation in non-treated section

Tb: Number of surviving worms before treatment in non-treated section

Tai: Number of surviving worms at observation in a treated section

It may be said that a test agent exhibiting a significantly highcontrolling value has the pesticidal activity. Preferably, it may bedetermined that a test agent having the controlling value of 30% or morehas substantial pesticidal activity, and it may be determined that atest agent having the controlling value of less than 30% has nosubstantial pesticidal activity.

The pesticidal agent in the present invention contains a chemicalsubstance having an ability to modulate the activity of insect cholineacetyltransferase or an agriculturally acceptable salt thereof as anactive ingredient.

In the present invention, an agriculturally acceptable salt refers to asalt in such a form that preparation of a controlling agent andapplication of the preparation do not become impossible, and may be asalt in any form. Specifically, examples of the salt include acidaddition salts with mineral acids such as hydrochloric acid, hydrobromicacid, hydriodic acid, sulfuric acid, nitric acid, and phosphoric,organic acids such as formic acid, acetic acid, propionic acid, oxalicacid, malonic acid, succinic acid, fumaric acid, maleic acid, lacticacid, malic acid, tartaric acid, citric acid, methanesulfonic acid, andethansulfonic acid, or acidic amino acids such as aspartic acid andglutamic acid; salts with inorganic bases such as sodium, potassium,magnesium, and aluminum, organic bases such as methylamine, ethylamine,and ethanolamine, or basic amino acids with lysine and ornithine; and anammonium salts.

In the present invention, the “pesticidal agent which comprises asubstance having an ability to modulate the activity of an insectcholine acetyltransferase or a an agriculturally acceptable salt thereofas an active ingredient” means an agent which can control pests bycontaining a substance having an ability to modulate the activity ofinsect choline acetyltransferase identified in the measuring method oran agriculturally acceptable salt thereof as an active ingredient.Preferable examples of the substance include a compound having anability to inhibit a reaction of a choline acetyltransferase withacetyl-CoA and choline. More preferable examples of the substanceinclude a substance having an ability to inhibit the reaction of theinsect choline acetyltransferase with acetyl-CoA and choline in acell-free system, wherein in the presence of the substance of 10 μM ormore the activity of the choline acetyltransferase is lower than that inthe absence of the substance. In addition, further preferable examplesof the substance include a substance having an ability to inhibit areaction of the insect choline acetyltransferase with acetyl-CoA andcholine in a cell-free system with an IC₅₀ of 100 μM or less.

In the present invention, the “method for assaying pesticidal activityof a test substance, which comprises a first step of measuring theactivity of a choline acetyltransferase selected from the group A in areaction system in which the choline acetyltransferase contacts with atest substance, and a second step of evaluating the pesticidal activityof the test substance based on the difference obtained by comparing theactivity measured in the first step with the activity of a control”indicates a method characterized by comprising the first step and thesecond step in various methods for assaying a pesticidal ability of atest substance.

Herein, the group A indicates:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 1;

(b) a protein comprising an amino acid sequence with deletion, additionor substitution of one or more amino acids in the amino acid sequence ofSEQ ID NO: 1, wherein said protein has choline acetyltransferaseactivity;

(c) a protein comprising an amino acid sequence that has sequenceidentity of 50% or more to the amino acid sequence of SEQ ID NO: 1,wherein said protein has choline acetyltransferase activity;

(d) a protein comprising an amino acid sequence that has sequencesimilarity of 75% or more to the amino acid sequence of SEQ ID NO: 1,wherein said protein has choline acetyltransferase activity;

(e) a protein comprising the amino acid sequence encoded by thenucleotide sequence of SEQ ID NO: 2 or 3;

(f) a protein comprising an amino acid sequence encoded by a nucleotidesequence that has sequence identity of 50% or more to the nucleotidesequence of SEQ ID NO: 2 or 3, wherein said protein has cholineacetyltransferase activity;

(g) a protein comprising an amino acid sequence encoded by apolynucleotide, wherein said polynucleotide hybridizes under a stringentcondition to a polynucleotide comprising a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 2 or 3, andwherein said protein has choline acetyltransferase activity;

(h) a protein comprising an amino acid sequence of an insect cholineacetyltransferase; and

(i) a protein comprising an amino acid sequence of a cotton aphidcholine acetyltransferase.

The first step is a step of measuring the activity of a cholineacetyltransferase in the state where a choline acetyltransferase iscontacted with a test substance by adding the test substance to theaforementioned various choline acetyltransferase activity measuringreaction systems.

The second step is a step of comparing the activity at measurement of atest substance with the substance of a control, and evaluating apesticidal ability based on the difference. Herein, a control means, forexample, in the case where a test substance dissolved in a solvent isadded to the reaction system, a test section in which only a solventsame as that used to dissolve the test substance is added.

A choline acetyltransferase used in a method for assaying a pesticidalability possessed by a test substance, having the first step and thesecond step, is a protein shown in the group A. Among proteins of thegroup A, a difference which can be recognized between an amino acidsequence of protein represented by (a) and amino acid sequences ofproteins represented by (b), (c), (e), (f), (g), (h) and (i) isdeletion, substitution, addition or the like of a part of amino acids.These include, for example, deletion due to processing which the proteinhaving an amino acid sequence represented by (a) undergoes in a cell. Inaddition, examples include deletion, substitution, addition and the likeof an amino acid generated by naturally occurring gene mutation due to aspices difference or an individual difference of an organism from whichthe protein is derived, or gene mutation which is artificiallyintroduced by a site-directed mutagenesis, a random mutagenesis,mutation treatment or the like.

The number of amino acids undergoing the deletion, substitution,addition or the like may be the number in a range that the cholineacetyltransferase activity of a choline acetyltransferase can be foundout. In addition, examples of substitution of an amino acid includesubstitution with an amino acid which is similar in characteristic inhydrophobicity, charge, pH and steric structure. Specific examples ofthe substitution include substitution in an group of (1) glycine,alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamicacid, asparagine, glutamine, (4) serine, threonine; (5) lysine,arginine; (6) phenylalanine, tyrosine and the like.

Examples of a procedure of artificially introducing the deletion,addition or substitution of an amino acid (hereinafter, collectivelyreferred to as alteration of amino acid in some cases) include aprocedure of introducing site-directed mutation into a DNA encoding anamino acid sequence represented by (a) and, thereafter, expressing thisDNA by a conventional method. Herein, examples of a site-directedmutagenesis include a method utilizing amber mutation (gapped duplexmethod, Nucleic Acids Res., 12, 9441-9456 (1984)), a method by PCR usingprimers for mutation introduction, and the like. In addition, examplesof a procedure of artificially altering an amino acid include aprocedure of randomly introducing mutation into a DNA encoding an aminoacid sequence represented by (a) and, thereafter, expressing this DNA bya conventional method. Herein, examples of a method of randomlyintroducing mutation include a method of performing PCR using a DNAencoding any of the aforementioned amino acid sequences as a template,and using a primer pair which can amplify each full length DNA atreaction condition under which an addition amount of each of dATP, dTTP,dGTP and dCTP used as substrates is changed from a conventionalconcentration, or at reaction condition under which a concentration ofMg²⁺ promoting a polymerase reaction is increased from a conventionalconcentration. Examples of the procedure of PCR include a methoddescribed, for example, in Method in Molecular Biology, (31), 1994,97-112. Another example includes a method described in WO 0009682.

As used herein, the “sequence identity” means the identity between twonucleotide sequences or two amino acid sequences. The “sequenceidentity” is determined by comparing the two sequences in an optimalalignment across the entire region of the sequence under comparison. Theoptimal alignment of the nucleotide sequence or amino acid sequenceunder comparison may allow for additions or deletions (for example,gaps). The sequence identity may be calculated by analyzing homologyusing programs such as FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci.USA. 4, 2444-2448 (1998)], BLAST [Altschul et al. Journal of MolecularBiology, 215, 403-410 (1990)], and CLUSTAL W [Thompson, Higgins &Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] to prepare analignment. These programs are available on the world wide web atddbj.nig.ac.jp, from the website of the DNA Data Bank of Japan [theinternational DNA data bank managed by the Center for InformationBiology and DNA Data Bank of Japan; CIB/DDBJ]. The sequence identity mayalso be analyzed using commercially available sequence analysissoftware. In particular, the sequence identity may be calculated byanalyzing homology using GENETYX-WIN Ver. 5 (manufactured by SoftwareDevelopment Co., Ltd.) by the Lipman-Pearson method (Lipman, D. J. andPearson, W. R., Science, 227, 1435-1441, (1985)) to prepare analignment.

When two optimally aligned amino acid sequences as described above havea difference in sequence as a result of conservative amino acidsubstitution, the “sequence similarity” is used in order to express theconservation of substituted amino acids. It may be said that thesequence similarity exists between sequence pairs which have differencesin sequence resulting from conservative amino acid substitutions. Thistype of sequence similarity can be analyzed using programs such as FASTAabove. Amino acids may be divided into four groups of hydrophobic aminoacids, neutral amino acids, acidic amino acids and basic amino acids.The substitution of an amino acid by another amino acid of the samegroup is termed conservative amino acid substitution.

Group of hydrophobic amino acids includes alanine (A), valine (V),leucine (L), isoleucine (I), methionine (M), tryptophan (W),phenylalanine (F) and proline (P).

Group of neutral amino acids includes glycine (G), serine (S), threonine(T), cysteine (C), tyrosine (Y), asparagine (N) and glutamine (Q).

Group of acidic amino acids includes aspartic acid (D) and glutamic acid(E).

Group of basic amino acids includes lysine (K), histidine (H) andarginine (R).

Examples of the “stringent condition” described in (g) include conditionunder which, in hybridization performed according to a conventionalmethod described in Sambrook J., Frisch E. F., Maniatis T., MolecularCloning 2nd edition, Cold Spring Harbor Laboratory press, for example, ahybrid is formed at 45° C. in a solution containing 6×SSC (a solutioncontaining 1.5 m NaCl and 0.15 m trisodium citrate is 10×SSC) and,thereafter, this is washed with 2×SSC at 50° C. (Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6). A salt concentration in awashing step can be selected from condition from 2×SSC (low stringentcondition) to 0.2×SSC (high stringent condition). A temperature in awashing step can be selected, for example, from condition from roomtemperature (low stringent condition) to 65° C. (high stringentcondition). Alternatively, both of a salt concentration and atemperature can be changed.

A protein described in (i) indicates a choline acetyltransferasepresents in a cotton aphid among an insect choline acetyltransferase,and includes a protein comprising the amino acid sequence described in(a).

While proteins of the group A include a protein described in (c) whichcomprises an amino acid sequence that has sequence identity of 50% ormore to the amino acid sequence of SEQ ID NO: 1 and which has cholineacetyltransferase activity, a protein having choline acetyltransferaseactivity and comprising an amino acid sequence that has sequenceidentity of at least 55, 60, 65, 70, 75 or 80% to the amino acidsequence of SEQ ID NO: 1 may be preferably used, and a protein havingcholine acetyltransferase activity and comprising an amino acid sequencethat has sequence identity of at least 85, 90 or 95% to the amino acidsequence of SEQ ID NO: 1 may be highly preferred.

While proteins of the group A also include a protein described in (d)which comprises an amino acid sequence that has sequence similarity of75% or more to the amino acid sequence of SEQ ID NO: 1 and which hascholine acetyltransferase activity, a protein having cholineacetyltransferase activity and comprising an amino acid sequence thathas sequence similarity of at least 80% to the amino acid sequence ofSEQ ID NO: 1 may be preferably used, and a protein having cholineacetyltransferase activity and comprising an amino acid sequence thathas sequence similarity of at least 85, 90 or 95% to the amino acidsequence of SEQ ID NO: 1 may be highly preferred.

A substance having pesticidal ability can be screened by using a methodof assaying pesticidal ability by measuring pesticidal ability orcontrolling effect on the aforementioned pests.

Alternatively, a substance having pesticidal ability can be alsoscreened by the aforementioned method of assaying pesticidal abilityusing a choline acetyltransferase. Specifically, when it has beenidentified that pesticidal ability of a test substance is a certainvalue or more, or a certain value or less by the aforementioned methodof assaying pesticidal ability using a choline acetyltransferase, asubstance having pesticidal ability can be screened by selecting thesubstance.

Since a substance selected by the screening method has pesticidalability, it can be used as a pesticidal agent containing the substanceor an agriculturally acceptable salt thereof as an active ingredient.

Control of pests can be usually performed by application of an effectiveamount of the pesticidal agent to a crop to be protected, a pest, or ahabitat of a pest.

When the pesticidal agent is used for agriculture and forestry, itsapplication amount is usually 0.1 to 1000 g in terms of an amount of thepesticidal agent per 1000 m². When the pesticidal agent is formulatedinto an emulsion, a water-dispersible powder, a flowable preparation, amicrocapsule preparation or the like, the agent is usually applied bydiluting with water to an active ingredient concentration of 1 to 10,000ppm, and spraying this and, when the pesticidal agent is formulated intoa granule, a powder or the like, the agent is usually applied as it is.

The pesticidal agent can be used by foliage-treating a plant such as acrop and the like which should be protected from pests, and can be alsoused by treating a seedbed before a plantlet of a crop is transplanted,or a planting hole or a strain base at planting. Further, for thepurpose of controlling pests habiting a soil of a cultivating land, theagent may be used by treating the soil. Alternatively, the agent may beused by a method of winding a resin preparation which has been processedto a sheet or a string, on a crop, stretching the preparation near acrop and/or spreading on a soil surface of a strain base.

When the pesticidal agent is used as a pest controlling agent forpreventing an epidemic, an emulsion, a water-dispersible powder, aflowable or the like is usually applied by diluting with water so thatan active ingredient concentration will become 0.01 to 10,000 ppm, andan oily agent, an aerosol, a fumigant, a poison bait or the like isapplied as it is.

Examples of one utility of the pesticidal agent include control of anexternal parasite of a livestock such as cattle, sheep, goat, andchicken, or a small animal such as dog, cat, rat, and mouse, in thiscase, the agent can be administered to an animal by the veterinarilyknown method. As a specific administration method, when systemic controlis intended, the agent is administered, for example, by a tablet, mixingin feed, suppository, injection (intramuscular, subcutaneous,intravenous, intraperitoneal etc.) and the like, when non-systemiccontrol is intended, the agent is used by a method of spraying an oilyagent or an aqueous liquid agent, performing pour on or spot ontreatment, washing an animal with a shampoo preparation or attaching aresin preparation which has been processed into a necklace or a ear tagto an animal. An amount of the pesticidal agent when administered to ananimal body is usually in a range of 0.1 to 1,000 mg as expressed bytotal amount of a compound A and a compound B per 1 kg of an animal.

An application amount and an application concentration of them may beboth different depending on the situations such as a kind of apreparation, an application time, an application place, an applicationmethod, a kind of a pest, a damage degree and the like, can be increasedor decreased regardless of the aforementioned range, and can beappropriately selected.

The aforementioned pesticidal agent can be used in the method ofcontrolling pests as described above.

In addition, a pest can be also controlled by identifying a substancehaving a pesticidal ability evaluated by the aforementioned method ofassaying pesticidal ability possessed by a pest substance, comprising afirst step and a second step using a choline acetyltransferase selectedfrom group A, and contacting the identified substance having pesticidalability with a pest. Herein, for contacting an identified substancehaving pesticidal ability with a pest, the aforementioned preparationmethod, application method and the like can be used.

An amino acid sequence shown in the group B is an amino acid sequence ofinsect choline acetyltransferase comprising any amino acid sequence ofthe following (a) to (h):

(a) the amino acid sequence of SEQ ID NO: 1;

(b) an amino acid sequence with deletion, addition or substitution ofone or more amino acids in the amino acid sequence of SEQ ID NO: 1,wherein said amino acid sequence has choline acetyltransferase activity;

(c) an amino acid sequence that has sequence identity of 50% or more tothe amino acid sequence of SEQ ID NO: 1, wherein said amino acidsequence has choline acetyltransferase activity;

(d) an amino acid sequence that has sequence similarity of 75% or moreto the amino acid sequence of SEQ ID NO: 1, wherein said amino acidsequence has choline acetyltransferase activity;

(e) the amino acid sequence encoded by the nucleotide sequence of SEQ IDNO: 2 or 3;

(f) an amino acid sequence encoded by a nucleotide sequence that hassequence identity of 50% or more to the nucleotide sequence of SEQ IDNO: 2 or 3, wherein said amino acid sequence has cholineacetyltransferase activity;

(g) an amino acid sequence encoded by a polynucleotide, wherein saidpolynucleotide hybridizes under a stringent condition to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 2 or 3, wherein said amino acidsequence has choline acetyltransferase activity; and

(h) an amino acid sequence of a cotton aphid choline acetyltransferase.

Among amino acid sequences of the group B, a difference which can berecognized between an amino acid sequence represented by (a) and aminoacid sequences represented by (b), (c), (e), (f), (g) and (h) isdeletion, substitution, addition or the like of a part of amino acids.These include, for example, deletion due to processing which the proteinhaving an amino acid sequence represented by (a) undergoes in a cell. Inaddition, examples include deletion, substitution, addition and the likeof an amino acid generated by naturally occurring gene mutation due to aspices difference or an individual difference of an organism from whichthe protein is derived, or gene mutation which is artificiallyintroduced by a site-directed mutagenesis, a random mutagenesis,mutation treatment or the like.

The number of amino acids undergoing the deletion, substitution,addition or the like may be the number in a range that the peptidaseactivity of a choline acetyltransferase can be found out. In addition,examples of substitution of an amino acid include substitution with anamino acid which is similar in characteristic in hydrophobicity, charge,pH and steric structure. Specific examples of the substitution includesubstitution in an group of (1) glycine, alanine; (2) valine,isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine,glutamine, (4) serine, threonine; (5) lysine, arginine; (6)phenylalanine, tyrosine and the like.

Examples of a procedure of artificially introducing the deletion,addition or substitution of an amino acid (hereinafter, collectivelyreferred to as alteration of amino acid in some cases) include aprocedure of introducing site-directed mutation into a DNA encoding anamino acid sequence represented by (a) and, thereafter, expressing thisDNA by a conventional method. Herein, examples of a site-directedmutagenesis include a method utilizing amber mutation (gapped duplexmethod, Nucleic Acids Res., 12, 9441-9456 (1984)), a method by PCR usingprimers for mutation introduction, and the like. In addition, examplesof a procedure of artificially altering an amino acid include aprocedure of randomly introducing mutation into a DNA encoding an aminoacid sequence represented by (a) and, thereafter, expressing this DNA bya conventional method. Herein, examples of a method of randomlyintroducing mutation include a method of performing PCR using a DNAencoding any of the aforementioned amino acid sequences as a template,and using a primer pair which can amplify each full length DNA atreaction condition under which an addition amount of each of dATP, dTTP,dGTP and dCTP used as substrates is changed from a conventionalconcentration, or at reaction condition under which a concentration ofMg²⁺ promoting a polymerase reaction is increased from a conventionalconcentration. Examples of the procedure of PCR include a methoddescribed, for example, in Method in Molecular Biology, (31), 1994,97-112. Another example includes a method described in WO 0009682.

As used herein, the “sequence identity” means the identity between twonucleotide sequences or two amino acid sequences. The “sequenceidentity” is determined by comparing the two sequences in an optimalalignment across the entire region of the sequence under comparison. Theoptimal alignment of the nucleotide sequence or amino acid sequenceunder comparison may allow for additions or deletions (for example,gaps). The sequence identity may be calculated by analyzing homologyusing programs such as FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci.USA. 4, 2444-2448 (1998)], BLAST [Altschul et al. Journal of MolecularBiology, 215, 403-410 (1990)], and CLUSTAL W [Thompson, Higgins &Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] to prepare analignment. These programs are available on the world wide web atddbj.nig.ac.jp, from the website of the DNA Data Bank of Japan [theinternational DNA data bank managed by the Center for InformationBiology and DNA Data Bank of Japan; CIB/DDBJ]. The sequence identity mayalso be analyzed using commercially available sequence analysissoftware. In particular, the sequence identity may be calculated byanalyzing homology using GENETYX-WIN Ver. 5 (manufactured by SoftwareDevelopment Co., Ltd.) by the Lipman-Pearson method (Lipman, D. J. andPearson, W. R., Science, 227, 1435-1441, (1985)) to prepare analignment.

When two optimally aligned amino acid sequences as described above havea difference in sequence as a result of conservative amino acidsubstitution, the “sequence similarity” is used in order to express theconservation of substituted amino acids. It may be said that thesequence similarity exists between sequence pairs which have differencesin sequence resulting from conservative amino acid substitutions. Thistype of sequence similarity can be analyzed using programs such as FASTAabove. Amino acids may be divided into four groups of hydrophobic aminoacids, neutral amino acids, acidic amino acids and basic amino acids.The substitution of an amino acid by another amino acid of the samegroup is termed conservative amino acid substitution.

Group of hydrophobic amino acids includes alanine (A), valine (V),leucine (L), isoleucine (I), methionine (M), tryptophan (W),phenylalanine (F) and proline (P).

Group of neutral amino acids includes glycine (G), serine (S), threonine(T), cysteine (C), tyrosine (Y), asparagine (N) and glutamine (Q).

Group of acidic amino acids includes aspartic acid (D) and glutamic acid(E).

Group of basic amino acids includes lysine (K), histidine (H) andarginine (R).

Examples of the “stringent condition” described in (g) include conditionunder which, in hybridization performed according to a conventionalmethod described in Sambrook J., Frisch E. F., Maniatis T., MolecularCloning 2nd edition, Cold Spring Harbor Laboratory press, for example, ahybrid is formed at 45° C. in a solution containing 6×SSC (a solutioncontaining 1.5 m NaCl and 0.15 m trisodium citrate is 10×SSC) and,thereafter, this is washed with 2×SSC at 50° C. (Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6). A salt concentration in awashing step can be selected from condition from 2×SSC (low stringentcondition) to 2×SSC (high stringent condition). A temperature in awashing step can be selected, for example, from condition from roomtemperature (low stringent condition) to 65° C. (high stringentcondition). Alternatively, both of a salt concentration and atemperature can be changed.

A protein having an amino acid sequence described in (h) indicates acholine acetyltransferase presents in a cotton aphid among an insectcholine acetyltransferase, and includes a protein comprising an aminoacid sequence described in (a).

While amino acid sequences of the group B include an amino acid sequencedescribed in (c) which has sequence identity of 50% or more to the aminoacid sequence of SEQ ID NO: 1 and which has choline acetyltransferaseactivity, an amino acid sequence having choline acetyltransferaseactivity and having sequence identity of at least 55, 60, 65, 70, 75 or80% to the amino acid sequence of SEQ ID NO: 1 may be preferably used,and an amino acid sequence having choline acetyltransferase activity andhaving sequence identity of at least 85, 90 or 95% to the amino acidsequence of SEQ ID NO: 1 may be highly preferred.

While amino acid sequences of the group B also include an amino acidsequence described in (d) which has sequence similarity of 75% or moreto the amino acid sequence of SEQ ID NO: 1 and which has cholineacetyltransferase activity, an amino acid sequence having cholineacetyltransferase activity and having sequence similarity of at least80% to the amino acid sequence of SEQ ID NO: 1 may be preferably used,and an amino acid sequence having choline acetyltransferase activity andhaving sequence similarity of at least 85, 90 or 95% to the amino acidsequence of SEQ ID NO: 1 may be highly preferred.

A protein having an amino acid sequence shown in the group B can beprepared, for example, according to a method described later using apolynucleotide encoding an amino acid sequence shown in the group B.

An insect choline acetyltransferase can be used as a reagent thatprovides an indicator to evaluate pesticidal activity. Specifically, forexample, an insect choline acetyltransferase can be used as a reagentthat provides an indicator to evaluate pesticidal activity by using as acholine acetyltransferase used in the method of assaying a pesticidalability using a choline acetyltransferase. In addition, a more specificmethod can be performed according to the aforementioned method ofmeasuring the activity of a choline acetyltransferase.

In addition, when an insect choline acetyltransferase is used as areagent that provides an indicator to evaluate a pesticidal activity, itis preferable that an insect choline acetyltransferase is a cholineacetyltransferase having an amino acid sequence selected from the groupB.

A polynucleotide having a nucleotide sequence encoding an amino acidsequence shown in the group B (hereinafter, referred to aspolynucleotide group B in some cases) has a nucleotide sequence fromwhich a protein having an amino acid sequence shown in the group B canbe produced, in a cell of an organism or an in vitro translation system.A polynucleotide group B may be DNA cloned from a nature, DNA in whichdeletion, substitution or addition of a nucleotide is introduced intoDNA cloned from a nature, for example, by a site-directed mutagenesis ora random mutagenesis, or an artificially synthesized DNA. Specifically,examples include a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 2.

<First Obtaining Method>

For example, a method of obtaining a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 3 will be shown below. Thepolynucleotide contains a polynucleotide comprising the nucleotidesequence of SEQ ID NO: 2 and is included in the polynucleotide group B.The method comprises obtaining total RNA from cotton aphids,synthesizing cDNA library from the RNA, and PCR amplification to obtainthe polynucleotide of interest.

A population of adults and larvae of Aphis gossypii, which have beenreared on leaves of potted cucumber, is scraped from the surface of theleaves with a small brush, and 630 mg of the obtained population iscrushed into a powder in liquid nitrogen using a mortar and a pestle.From the resulting frozen crushed powder, RNA is isolated using a RNAextracting reagent ISOGEN (manufactured by Nippon Gene) as follows.After 10 ml of ISOGEN is added to the frozen crushed powder in themortar, the crushed powder is ground for 10 minutes while kept on ice.After grinding, a fluid sample is transferred to a 15 ml tube with apipette, and 2 ml of chloroform (manufactured by Wako Pure ChemicalIndustries, Ltd.) is added thereto. Immediately, the mixture isvigorously shaken for 15 seconds and then left at rest at roomtemperature for 3 minutes. Then, the resulting mixture is centrifuged at12,000×g at 4° C. for 15 minutes, and each 5 ml of aqueous layer aretransferred to two new tubes. After 5 ml of ISOGEN is added to eachtube, the mixture was immediately shaken vigorously for 15 seconds, andleft at rest at room temperature for 3 minutes. Then, the resultingmixture is centrifuged at 12,000×g at 4° C. for 15 minutes, and each 10ml of aqueous layer are transferred to new 50 ml tubes, respectively.Subsequently, 10 ml of isopropanol (manufactured by Wako Pure ChemicalIndustries, Ltd.) is added to each tube, and the mixture is kept on icefor 30 minutes. The resulting mixture is centrifuged at 12,000×g at 4°C. for 10 minutes to precipitate RNA. After the supernatant is removed,20 ml of 70% ethanol is added to the residue. The resulting mixture iscentrifuged at 10,000×g at 4° C. for 5 minutes. After the supernatant isremoved, the precipitate of total RNA is slightly dried and thendissolved in 1 ml of commercially available RNase-free water (NacalaiTesque, Inc.). An absorbance of the prepared total RNA is measured at260 nm to calculate a concentration according to a conventional method.

RT-PCR is performed employing total RNA of cotton aphid obtained by theaforementioned method as a template, and using random primers(manufactured by Invitrogen) and superscript III (manufactured byInvitrogen) according to the manual annexed to the reagent, tosynthesized a first-strand cDNA.

PCR is performed employing cDNA library of cotton aphid obtained by theaforementioned method as a template, and using an oligonucleotide primercomprising the nucleotide sequence of SEQ ID NO: 4 and anoligonucleotide primer comprising the nucleotide sequence of SEQ ID NO:5 as well as Pfu Ultra HF Taq polymerase (manufactured by Stratagene)according to the manual annexed to the reagent. The PCR conditions areas follows: an initial denaturation at 94° C. for 10 minutes; followedby 35 cycles of PCR, one cycle being 94° C. for 20 seconds, 53° C. for20 seconds, and 72° C. for 3 minutes; followed by 72° C. for 7 minutes.

As described above, a polynucleotide comprising the nucleotide sequenceof SEQ ID NO: 3 can be obtained.

<Second Obtaining Method>

A polynucleotide shown in the polynucleotide group B can be alsoobtained by preparing a polynucleotide with mutation introduced thereinby a method utilizing amber mutation which is the aforementionedsite-directed mutagenesis, a method by PCR using a primer forintroducing mutation or the like, using as a template a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 2 or 3.

<Third Obtaining Method>

A polynucleotide shown in the polynucleotide group B can be alsoobtained by a hybridization method using a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 2 or 3 as a probe. More specifically,the third obtaining method can be performed according to theaforementioned conventional hybridization method described in SambrookJ., Frisch E. F., Maniatis T., Molecular Cloning 2nd edition, publishedby Cold Spring Harbor Laboratory press.

<Fourth Obtaining Method>

Alternatively, a polynucleotide shown in the polynucleotide group B canbe also obtained by preparing a primer based on an amino acid sequenceof the known insect choline acetyltransferase and performing PCR. Forisolation of homologues of choline acetyltransferase gene from otherinsect species such as German cockroach (Blatella germanica), degenerateprimers are designed using Codehop program (publicly accessible on thewebsite of Blocks Protein Analysis Server operated within the FredHutchinson Cancer Research Center atblocks.fhcrc.org/blocks/codehop.html), and based on the sequence of theaforementioned cotton aphid choline acetyltransferase gene and thepreviously-known amino acid sequences from such as D. melanogaster (NCBIaccession number P07668), Ceanorhabditis elegans (P32756) and Anophelesgambiae (XP_(—)312586).

Partial sequences of a homologue of choline acetyltransferase gene of aselected insect species are amplified by a series of PCR usingfirst-strand cDNA derived from the insect species as a template. Herein,the first-strand cDNA as a template is prepared by the aforementionedmethod using Superscript III. Amplification by PCR is performed using aset of degenerate primers as a forward primer and a reverse primer aswell as Amplitaq Gold (manufactured by Applied Biosystems) according tothe manufacturer's procedure annexed to the reagent. The PCR conditionsare those for touchdown PCR as follows: an initial denaturation at 94°C. for 10 minutes; followed by 10 cycles of touchdown-PCR, one cyclebeing 94° C. for 30 seconds, 60° C. for 1 minute with a decrease of 1°C. per cycle, and 72° C. for 1 minute and 30 seconds; followed by 25cycles of PCR, one cycle being 94° C. for 30 seconds, 50° C. for 1minute, and 72° C. for 1 minute and 30 seconds; and followed by 72° C.for 7 minutes. The PCR product is analyzed and purified by agarose gelelectrophoresis to obtain DNA of interest. Further, the obtained DNA iscloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

Then, primers specific for the resulting partial sequences of the insecthomologue of choline acetyltransferase gene are designed, and 3′RACE PCRor 5′RACE PCR is performed in order to obtain a full-length sequence ofthe gene. The 3′RACE PCR is performed employing first-strand cDNAprepared from the insect total RNA as a template and using SMART PCRcDNA Synthesis Kit (manufactured by Clontech) according to themanufacturer's instructions annexed to the kit. The 5′RACE PCR isperformed employing first-strand cDNA prepared from the insect total RNAas a template and using 5′/3′ RACE Kit, 2nd Generation (manufactured byRoche) according to the manufacturer's instructions annexed to the kit.

In 3′RACE reaction, a forward primer specific for the sequence ofinterest is used in combination with universal primer mix (UPM)contained in SMART PCR cDNA Synthesis Kit as a reverse primer. The

PCR conditions are those for touchdown PCR as follows: an initialdenaturation at 94° C. for 10 minutes; followed by 10 cycles oftouchdown-PCR, one cycle being 94° C. for 20 seconds, 60° C. for 20seconds with a decrease of 1° C. per cycle, and 72° C. for 2 minutes;followed by 25 cycles of PCR, one cycle being 94° C. for 20 seconds, 50°C. for 20 seconds, and 72° C. for 3 minutes; and followed by 72° C. for7 minutes. The resulting PCR product is analyzed and purified by agarosegel electrophoresis to obtain DNA of interest. Further, the obtained DNAis cloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

When a distinct amplification product is not obtained by the first-roundPCR, nested PCR is performed using the first-round PCR product as atemplate. As primers, a specific forward primer which is designed tobind to internal sequence of the first-round PCR product is used incombination with NUP primer contained in SMART PCR cDNA Synthesis Kit asa reverse primer. The PCR conditions are as follows: an initialdenaturation at 95° C. for 10 minutes; followed by 35 cycles of PCR, onecycle being 95° C. for 20 seconds, 50° C. for 20 seconds, and 72° C. for3 minutes; followed by 72° C. for 7 minutes. The resulting PCR productis analyzed and purified by agarose gel electrophoresis to obtain DNA ofinterest. Further, the obtained DNA is cloned into the pCR4-TOPO vector(manufactured by Invitrogen), and sequenced.

In 5′RACE reaction, a reverse primer specific for the sequence ofinterest is used in combination with Oligo-d(T)-anchor primer1 containedin 5′/3′RACE Kit, 2nd Generation as a forward primer. The PCR conditionsare those for touchdown PCR as follows: an initial denaturation at 94°C. for 10 minutes; followed by 10 cycles of touchdown-PCR, one cyclebeing 94° C. for 30 seconds, 58° C. for 30 seconds with a decrease of 1°C. per cycle, and 72° C. for 2 minutes; followed by 25 cycles of PCR,one cycle being 94° C. for 30 seconds, 48° C. for 1 minute, and 72° C.for 2 minutes; and followed by 72° C. for 7 minutes. The resulting PCRproduct is analyzed and purified by agarose gel electrophoresis toobtain DNA of interest. Further, the obtained DNA is cloned into thepCR4-TOPO vector (manufactured by Invitrogen), and sequenced.

When a distinct amplification product is not obtained by the first-roundPCR, nested PCR is performed using the first-round PCR product as atemplate. As primers, a specific reverse primer which is designed tobind to internal sequence of the first-round PCR product is used incombination with PCR Anchor primer contained in 5′/3′RACE Kit, 2ndGeneration as a forward primer. The PCR conditions are as follows: aninitial denaturation at 94° C. for 10 minutes; followed by 10 cycles ofPCR, one cycle being 94° C. for 15 seconds, 55° C. for 30 seconds, and72° C. for 40 seconds; followed by 25 cycles of PCR, one cycle being 94°C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 40 secondsextended with 20 seconds each cycle; followed by 72° C. for 7 minutes.The resulting PCR product is analyzed and purified by agarose gelelectrophoresis to obtain DNA of interest. Further, the obtained DNA iscloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

The above sequencing results reveal 5′-terminal sequence and 3′-terminalsequence, each encoding N-terminal region and C-terminal region of theinsect choline acetyltransferase, respectively.

Thus, a polynucleotide shown in the polynucleotide group B can beobtained by PCR by preparing primers based on amino acid sequences ofthe known insect choline acetyltransferases.

A polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence of the polynucleotide group B can be used forobtaining a polynucleotide shown in the polynucleotide group B using ahybridization method.

The obtaining method in the present invention comprises a step ofdetecting a desired polynucleotide by hybridization, a step ofidentifying the detected desired polynucleotide, and a step ofrecovering the identified desired polynucleotide. Each step will beexplained specifically below.

A step of detecting a desired polynucleotide by hybridization, and astep of identifying the detected desired polynucleotide can be performedby using, as a probe, a polynucleotide having a nucleotide sequencehaving complementarity to a nucleotide sequence of a polynucleotidegroup B, according to the method described, for example, in “MolecularCloning: A Laboratory Manual 2nd edition” (1989), Cold Spring HarborLaboratory Press, “Current Protocols In Molecular Biology” (1987), JohnWiley & Sons, Inc. ISBN0-471-50338-X and the like.

Specifically, for example, a DNA comprising a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 2 is labeled witha radioisotope or a fluorescently labeled by the known method usingRandom Primed DNA Labelling Kit (manufactured by Boehringer), RandomPrimer DNA Labelling Kit Ver.2 (manufactured by TAKARA SHUZO Co., Ltd.),ECL Direct Nucleic Acid Labelling and Ditection System (manufactured byAmersham Biosciences), or Megaprime DNA-labelling system (manufacturedby Amersham Biosciences), and this can be used as probe.

Examples of condition for hybridization include stringent condition, andspecifically, examples include condition under which incubation isperformed at 65° C. in the presence of 6×SSC (0.9M NaCl, 0.09M sodiumcitrate), a 5×Denhart's solution (0.1% (w/v) Ficoll 400, 0.1% (w/v)polyvinylpyrrolidone, 0.1% BSA), 0.5% (w/v) SDS and 100 μg/ml denaturedsalmon spermatozoon DNA, or in a DIG EASY Hby solution (BoehringerMamnnheim) containing 100 μg/ml denatured salmon spermatozoon DNA, then,incubation is performed two times at room temperature for 15 minutes inthe presence of 1×SSC (0.15 m NaCl, 0.015 m sodium citrate) and 0.5% SDSand, further, incubation is performed at 68° C. for 30 minutes in thepresence of 0.1×SSC (0.015 m NaCl, 0.0015 m sodium citrate) and 0.5%SDS.

More specifically, for example, a probe labeled with ³²P can be made byemploying a polynucleotide comprising a nucleotide sequencecomplementary to a nucleotide sequence of a polynucleotide group B as atemplate, using Megaprime DNA-labelling system (manufactured by AmershamPharmacia Biotech) and using a reaction solution designated in a kit.Colony hybridization is performed using this probe according to aconventional method, incubation is performed at 65° C. in the presenceof 6×SSC (0.9M NaCl, 0.09M sodium citrate), a 5×Denhart's solution (0.1%(w/v) Ficoll 400, 0.1% (w/v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w/v)SDS and 100 μg/ml denatured salmon spermatozoon DNA, or in a DIG EASYHyb solution (Boehringer Mannheim) containing 100 μg/ml denatured salmonspermatozoon DNA, then, incubation is performed two times at roomtemperature for 15 minutes in the presence of 1×SSC (0.15 m NaCl, 0.015m sodium citrate) and 0.5% SDS and, further, incubation is performed at68° C. for 30 minutes in the presence of 0.1×SSC (0.015 m NaCl, 0.0015 msodium citrate) and 0.5% SDS, thereby, (a colony containing) ahybridizing polynucleotide can be detected. Thus, a desiredpolynucleotide can be detected by hybridization, and the detecteddesired polynucleotide can be identified.

For recovering the identified desired polynucleotide, a plasmid DNA canbe recovered from a colony containing the polynucleotide detected andidentified by the aforementioned method, for example, according to amethod such as the alkali method described in “Molecular Cloning: ALaboratory Manual 2nd edition” (1989), Cold Spring Harbor LaboratoryPress. A nucleotide sequence of the recovered desired polynucleotide(plasmid DNA) can be confirmed by a Maxam Gilbert method (described, forexample, in Maxam, A. M and W. Gilbert, Proc. Natl. Acad. Sci. USA, 74,560, 1977 etc.) or a Sanger method (described, for example, in Sanger,F. and A. R. Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, andNicklen and A. R. Coulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977etc.). Thereupon, for example, commercially available Termo Segenase IIdye terminator cycle sequencing kit (manufactured by Amershambiosciences), Dye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by Applied Biosystems) and the like can be used.

A polynucleotide comprising a partial nucleotide sequence of anucleotide sequence of the polynucleotide group B or a nucleotidesequence complementary to the partial nucleotide sequence can be usedfor obtaining a polynucleotide shown in the polynucleotide group B usingPCR. More specifically, examples include a polynucleotide comprising anucleotide sequence of SEQ ID NO: 4 or 5. The obtaining method in thepresent invention includes a step of amplifying a desired polynucleotideby PCR, a step of identifying the amplified desired polynucleotide, anda step of recovering the identified desired polynucleotide. Each stepwill be specifically explained below.

In a step of amplifying a desired polynucleotide by PCR, specifically, aDNA designed and synthesized from a partial nucleotide sequence of anucleotide sequence of a polynucleotide group B or a nucleotide sequencecomplementary to the partial nucleotide sequence, based on an about 20bp to about 40 bp nucleotide sequence, for example, a nucleotidesequence selected from a nucleotide sequence of SEQ ID NO: 2 and asequence complementary to the nucleotide sequence of SEQ ID NO: 2 can beused as a primer set. Examples of a primer set include a set of apolynucleotide comprising a nucleotide sequence represented by SEQ IDNO: 4 and a polynucleotide comprising the nucleotide sequence of SEQ IDNO: 5. A PCR reaction solution is prepared, for example, by adding areaction solution designated by a commercially available PCR kit to acDNA library prepared by the aforementioned method. Reaction conditioncan be changed depending on a primer set to be used, and for example,condition under which after incubation at 94° C. for 10 seconds, around40 cycles is repeated, 1 cycle being 94° C. for 15 seconds, 60° C. for15 seconds, and 72° C. for 3 minutes and, further, incubation isperformed at 72° C. for 3 minutes, condition under which incubation isperformed at 94° C. for 2 minutes, thereafter, incubation is performedat about 8° C. for 3 minutes and, thereafter, around 40 cycles isrepeated, 1 cycle being 94° C. for 30 seconds, 55° C. for 30 seconds,and 72° C. for 4 minutes, or condition under which 5 to 10 cycles isperformed, 1 cycle being incubation at 94° C. for 5 seconds and, then,72° C. for 4 minutes and, further, around 20 to 40 cycles is performed,1 cycle being incubation at 94° C. for 5 seconds and, then, 70° C. for 4minutes, can be used. In the PCR, for example, PfuUltra High Fidelitypolymerase (manufactured by Stratagene), Amplitaq Gold (manufactured byApplied Biosystems), Takara Heraculase (Trademark) (manufactured byTAKARA SHUZO Co., Ltd.), a DNA polymerase contained in Advantage cDNAPCR Kit (manufactured by Clonetech), TaKaRa Ex Taq (manufactured byTAKARA SHUZO Co., Ltd.), PLATINUM™ PCR SUPER Mix (manufactured byLifetech Oriental) can be used.

Identification of a desired polynucleotide amplified by PCR can beperformed by measuring a molecular weight by agarose gel electrophoresisaccording to the method described in “Molecular Cloning: A LaboratoryManual 2nd edition” (1989), Cold Spring Harbor Laboratory Press. Inaddition, regarding the amplified desired polynucleotide, a sequencingreaction is performed using a commercially available DNA sequencingreaction kit, for example, Dye Terminator Cycle Sequencing FS ReadyReaction Kit (manufactured by Applied Biosystems) according to a manualannexed to the kit, and the nucleotide is analyzed using a DNA sequencer3100 (manufactured by Applied Biosystems), thereby, a nucleotidesequence of the amplification fragment can be read.

Examples of a method of recovering the identified desired polynucleotideinclude a method of purifying and recovering the aforementionedpolynucleotide identified by agarose gel electrophoresis from an agarosegel according to the method described in “Molecular Cloning: ALaboratory Manual 2nd edition” (1989), Cold Spring Harbor LaboratoryPress. In addition, the thus recovered polynucleotide or a desiredpolynucleotide amplified by PCR can be cloned into a vector according toa conventional method described in “Molecular Cloning: A LaboratoryManual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, and“Current Protocols In Molecular Biology” (1987), John Wiley & Sons, Inc.ISBN0-471-50338-X. Examples of a vector to be used include pUCA119(manufactured by TAKARA SHUZO Co., Ltd.), pTVA118N (manufactured byTAKARA SHUZO Co., Ltd.), pBluescriptII (manufactured by Toyobo Co.,Ltd.), pCR2.1-TOPO (manufactured by Invitrogen) and the like. Inaddition, a nucleotide sequence of the cloned polynucleotide can beconfirmed by a Maxam Gilbert method (described, for example, in Maxam,A. M & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or aSanger method (described, for example, in Sanger, F. & A. R. Coulson, J.Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and A. R. Coulson.,Proc. Natl. Acad. Sci. USA, 74, 5463, 1977). Thereupon, for example, acommercially available Termo Segenase II dye terminator cycle sequencingkit (manufactured by Amersham biosciences), Dye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by Applied Biosystems)and the like can be used.

In addition, a polynucleotide having a partial nucleotide sequence of anucleotide sequence of the polynucleotide group B or a nucleotidesequence complementary to the partial nucleotide sequence can be usedfor obtaining a polynucleotide shown in the polynucleotide group B usingnot only a PCR method, but also the aforementioned hybridization method.More specifically, examples include a polynucleotide comprising anucleotide sequence represented by SEQ ID NO: 4 or 5.

Examples of a method for preparing a protein comprising an amino acidsequence shown in the group B include a method of culturing atransformant with a polynucleotide selected from a polynucleotide groupB introduced therein, and recovering the produced protein. In addition,for preparing a transformant used herein, it is a work such aspreparation of a circular polynucleotide containing a polynucleotide inwhich a polynucleotide selected from a polynucleotide group B isoperably ligated to a bacteriophage promoter. The method will beexplained in detail below.

In addition, a choline acetyltransferase shown in the group A which isused in the method of assaying a pesticidal activity using a cholineacetyltransferase can be prepared and obtained by the similar method,using a polynucleotide comprising a nucleotide sequence encoding thecholine acetyltransferase.

A bacteriophage promoter means a promoter of a gene contained in abacteriophage genome. Among them, examples of a promoter ofbacteriophage used for expressing a foreign gene include a promoter ofT7 RNA polymerase gene, T3 RNA polymerase gene and SP6 RNA polymerasegene.

In the present invention, “operably linked” means that a polynucleotidecontaining a gene of interest is linked downstream of a polynucleotidecontaining a promoter sequence so that the gene of interest can betranscribed in a used transcription system. Specifically, for example,when a promoter of T7 RNA polymerase gene described later is used, apolynucleotide containing a gene of interest may be linked downstream ofa promoter of T7 RNA polymerase gene. In addition, for example, when apromoter other than T7 RNA polymerase gene promoter is used, it is alsopossible to link a polynucleotide containing a gene of interestdownstream of a polynucleotide containing a promoter sequence other thanT7 RNA polymerase gene promoter. More specifically, for example, when aplasmid pET41a(+)(Novagen) vector utilizing T7 RNA polymerase genepromoter is used, the polynucleotide can be operably linked by ligatinga gene of interest into a restriction enzyme site such as NcoI, EcoRV,BamHI, EcoRI, StuI, PstI, SacI, SalI, HindIII, NotI, EagI and XhoIlocated downstream of T7 RNA polymerase gene promoter.

In the present invention, the “circular polynucleotide” is apolynucleotide which has been made to be circular by binding of ends ofthe polynucleotide strand, and examples include chromosomal DNAs of manybacteria in addition to a plasmid DNA, a bacmid DNA and the like.

A plasmid DNA is a relatively low-molecular circular polynucleotide, andexamples include pET (manufactured by Takara Mirus Bio Inc.) andpBluescriptII (manufactured by Stratagene), used for cloning andexpression in E. coli. Additional examples include pFastBac1, pFastBacHT A, pFastBac HT B, pFastBac HT C, pFastBac Dual, pBlueBacII(manufactured by Invitrogen), pAcSG2 (manufactured by Pharmingen) andthe like, which contain a baculovirus expression cassette.

The bacmid is a high molecular weight DNA that consists of a BAC(bacterial artificial chromosome) that contains the entire baculoviralgenome, for example bMON14272 (136 kb) that is present in DH10Bac™ E.coli cells (invitrogen). Bacmid DNA propagates as a large plasmid in E.coli cells and may contain an expression cassette for expression of aforeign gene under control of a baculoviral promoter.

A circular polynucleotide in which a polynucleotide comprising anucleotide sequence encoding an amino acid sequence shown in the group Bis operably linked to a bacteriophage promoter is specifically, forexample, a circular polynucleotide containing a DNA comprising a cottonaphid choline acetyltransferase gene operably linked to a bacteriophageT7 RNA polymerase promoter, and can be prepared and obtained, forexample, according to the following method.

DNA fragment containing the choline acetyltransferase gene is amplifiedby PCR, using a plasmid DNA containing a cotton aphid cholineacetyltransferase gene cloned in accordance with the aforementionedmethod as a template, with a primer specific to the cholineacetyltransferase gene to which a Bam HI restriction site is added and aprimer specific to the choline acetyltransferase gene to which a XhoIrestriction site is added. The resulting PCR products are cleaved withBam HI and XhoI, and the obtained approximately 2.2 kbp of DNA fragmentcontaining the cotton aphis choline acetyltransferase gene is ligated toa plasmid vector pET41a (+) (manufactured by Novagen) digested inadvance with Bam HI and XhoI. The plasmid obtained in this way is oneexample of circular polynucleotide containing DNA fragment comprisingthe cotton aphis choline acetyltransferase gene operably linked tobacteriophage T7 RNA polymerase promoter.

Similarly, a circular polynucleotide can be prepared by ligatingnucleotides encoding an amino acid sequence shown in the group B to avector.

In the present invention, the “origin of replication” is the specificDNA sequence necessary for replicating itself in a host cell. Examplesof origin of replication include colE1 and f1 for bacterial plasmids.

A transformant is a eukaryotic cell or a prokaryotic cell which has beengenetically altered by introduction of a foreign polynucleotide into acell. Examples of a transformant include an Escherichia coli celltransformed by introduction of a plasmid used for gene cloning or geneexpression in E. coli, such as pET (Novagen) or pBluescript II(Stratagene). In addition, examples of the technique of introducing aDNA into a host cell include transformation, transfection, protoplastfusion, lipofection, electroporation and the like.

Examples of a transformant in which a polynucleotide encoding an aminoacid sequence shown in the group B is introduced include transformedEscherichia coli in which a DNA comprising a cotton aphid cholineacetyltransferase gene operably linked to a bacteriophage T7 RNApolymerase promoter is introduced. Specifically, the transformant can beprepared according to the following method.

A transformant can be prepared by introducing into an Escherichia colicell a plasmid vector pET41a (+) (Novagen) in which a DNA containing acotton aphid choline acetyltransferase gene is inserted between Bam HIsite and Xho I site, according to the method described in “MolecularCloning: A Laboratory Manual 2nd edition” (1989), Cold Spring HarborLaboratory Press. Alternatively, a transformant can be also prepared bytransforming E. coli using the aforementioned plasmid DNA into which afragment containing a bacteriophage T7 RNA polymerase promoter and acotton aphid choline acetyltransferase gene is inserted, according to amethod described in a manual annexed to Escherichia coli BL21 (DE3)competent cells (Invitrogen).

A choline acetyltransferase can be prepared by culturing a transformantprepared by the aforementioned method, and recovering the producedinsect-derived choline acetyltransferase.

Choline acetyltransferase protein may be produced by a recombinant E.coli expression system. This system is the most frequently usedprokaryotic expression system for the high-level production ofheterologous proteins. E. coli is genetically and physiological the bestcharacterized organism known, it is easy to manipulate, many tools areavailable, it is able to grow very fast, it grows on cheap complex orwell-defined minimal media and it has an extremely high capacity tosynthetize heterologous protein.

Alternatively, choline acetyltransferase protein may be produced by forexample a recombinant baculovirus/Sf9 cell expression system. Thissystem is one of the most powerful and versatile eukaryotic expressionsystems available, and may be used to express heterologous genes frommany different sources, including fungi, plants, bacteria and viruses.

In addition, an insect-derived choline acetyltransferase produced byculturing a transformant is lysed by a method such as sonication, Frenchpress, and Dyno mill, and recovered in a form contained in a cell crudeextract, and a purified protein can be obtained by using a procedureconventionally used in enzyme purification such as ion exchange columnchromatography, reverse phase column chromatography, gel filtrationcolumn chromatography and the like. Alternatively, when it is devisedthat an insect-derived choline acetyltransferase is produced in a formwith His-tag, a purified protein can be obtained rapidly from a cellcrude extract by affinity column chromatography which specificallyrecognizes and binds to the His-tag. By the method, an insect-derivedcholine acetyltransferase can be prepared.

For example, an insect-derived choline acetyltransferase can be preparedby culturing a E. coli cell transformed with a DNA fragment containing acotton aphid choline acetyltransferase gene operably linked tobacteriophage T7 RNA polymerase promoter, and grinding the cell with aFrench press, followed by purification with column chromatography.

To be more concrete, for example, a recombinant E. coli containing theDNA fragment comprising the cotton aphid choline acetyltransferase geneoperably linked to the bacteriophage T7 RNA polymerase promoter, whichprepared by the above method, is rotary cultured overnight at 37° C.,250 rpm. In the next morning, the culture is diluted 1/100 in LB mediumcontaining 50 mg/L of Kanamycin, to which IPTG is added until a finalconcentration of 10 μM, and grown at 22° C., 60 rpm for 4 days toproduce recombinant choline acetyltransferase protein in the E. coli.The culture is centrifuged at 7,000 rpm for 10 minutes to collect the E.coli. To the collected E. coli, breaking buffer (0.1M Sodium phosphatebuffer pH 7.6, 1 tablet of Complete EDTA-free protease inhibitorcocktail (manufactured by Roch)) is added and suspended. Further, the E.coli is lysed with a pressure between 1,300 psi and 1,500 psi, usingFrench press (manufactured by Thermo Spectronic), in accordance with themethod described in the attached instruction. The French pressedsolution is centrifuged at 14,000 rpm, 2° C., for 60 minutes, and theresulting supernatant is filtrated through a 0.45 μm filter. The sampleis injected into HiTrap Chelating HP (manufactured by Amershambiosciences) column or HisTrap HP (manufactured by Amersham biosciences)column, which is equilibrated with His buffer A (0.1M Sodium phosphatebuffer pH 7.6, 10% glycerol). Afterwards, the column is washed with 5column volumes of a buffer, in which 95% of His buffer A and 5% of Hisbuffer B (0.1M Sodium phosphate buffer pH 7.6, 500 mM imidazole, 10%glycerol) are mixed. Next, the column is washed with 15 column volumesof a buffer, in which 90% of His buffer A and 10% of His buffer B aremixed. Next, the column is washed with 15 column volumes of a buffer, inwhich 85% of His buffer A and 15% of His buffer B are mixed. Next, thecolumn is washed with 15 column volumes of a buffer, in which 80% of Hisbuffer A and 20% of His buffer B are mixed. Afterwards, 15 columnvolumes of a buffer in which 60% of His buffer A and 40% of His buffer Bare mixed is prepared and injected into the column. One ml of aliquotsof the eluted fraction is pooled and an aliquot is analyzed withSDS-PAGE to determine fractions containing 115 kDa of cholineacetyltransferase. These fractions are the solution enriched in thetarget choline acetyltransferase.

An insect choline acetyltransferase comprising an amino acid sequenceshown in the group B can be used as a research tool. For example, it canbe used as a research tool for performing study such as assaying of thepesticidal ability, screening of a chemical substance having apesticidal ability, and the like. In addition, for example, also instudy of analyzing action and mechanism of an agent which acts on acholine acetyltransferase, a choline acetyltransferase can be utilizedas a research tool.

In addition, polynucleotides encoding amino acid sequences shown in thegroup B and polynucleotides having a nucleotide sequence havingcomplementarity to them, as well as partial nucleotide sequences ofpolynucleotides encoding amino acid sequences shown in the group B, orpolynucleotides having nucleotide sequences having complementarity tothe partial nucleotide sequences, and a polynucleotide complying anucleotide sequence represented by SEQ ID NO: 4 or 5 can be used as aresearch tool. For example, a part of them functions as a polynucleotideused in a method of preparing a choline acetyltransferase as describedabove. In addition, apart can be used as an important research tool forperforming obtaining a polynucleotide shown in a polynucleotide group Busing PCR, or obtaining a polynucleotide shown in a polynucleotide groupB using hybridization, as described above.

Particularly, upon implementation of screening of a pesticidal agent,they can be used as an experimental tool for an experiment which isperformed for screening. Specifically, they can be used as anexperimental tool for an experiment which is performed uponimplementation of the assaying of a pesticidal ability, screening of achemical substance having a pesticidal ability, and the like.

Further, the present invention also includes a system which comprises ameans to input, store and manage data information of an ability of testsubstances, wherein said ability is an ability to modulate the activityof an insect choline acetyltransferase (hereinafter, referred to asmeans a in some cases), a means to query and retrieve the datainformation based on a desired criterion (hereinafter, referred to asmeans b in some cases), and a means to display and output the resultwhich is queried and retrieved (hereinafter, referred to as means c insome cases) (hereinafter, referred to as present system in some cases).

First, a means a will be explained. A means a is a means to, after datainformation of an ability to modulate the activity of an insect-derivedcholine acetyltransferase possessed by the test substance is inputted,store and manage the inputted information, as described above. Theinformation is inputted by an inputting means 1, and is usuallymemorized in a memory means 2. Examples of an inputting means includemeans which can input the information such as a keyboard and a mouse.When inputting and storing or managing of the information are completed,a procedure progresses to a next means b. For storing or managing theinformation, a large amount of data may be effectively stored andmanaged by inputting information having a data structure using ahardware such as a computer, and a software such as OS and databasemanagement, and storing the information into a suitable memory device,for example, computer-readable recording medium such as a flexible disc,a photomagnetic disc, CD-ROM, DVD-ROM, and a hard disc.

A means b will be explained. A means b is a means to query and retrievethe data information stored and managed by a means of a based oncriterion for obtaining a desired result, as described above. For theinformation, when criterion for querying and retrieving is inputted byan inputting means 1, and information in conformity with the criterionis selected among the information usually memorized in a memory means 2,a procedure progresses to a next means c. The selected result is usuallymemorized in a memory means 2 and, further, can be displayed by adisplaying or outputting means 3.

A means c will be explained. A means c is a means to display and outputthe result which is queried and retrieved, as described above. Examplesof the displaying or outputting means 3 include a display, a printer andthe like, and the result may be displayed on a display device of acomputer, or may be outputted on a paper by printing.

EXAMPLES

The present invention will be explained in more detail below by way ofExamples, but the present invention is not limited to these particularExamples.

Example 1 Extraction of Total RNA from Cotton Aphid and German Cockroach

(1) Extraction of Total RNA from Cotton Aphid.

A population of adults and larvae of cotton aphid (Aphis gossypii),which had been reared on leaves of potted cucumber, was scraped from thesurface of the leaves with a small brush, and 630 mg of the obtainedpopulation was crushed into a powder in liquid nitrogen using a mortarand a pestle. From the resulting frozen crushed powder, RNA was isolatedusing a RNA extracting reagent ISOGEN (manufactured by Nippon Gene) asfollows. After 10 ml of ISOGEN was added to the frozen crushed powder inthe mortar, the crushed powder was ground for 10 minutes while kept onice. After grinding, a fluid sample was transferred to a 15 ml tube witha pipette, and 2 ml of chloroform (manufactured by Wako Pure ChemicalIndustries, Ltd.) was added thereto. Immediately, the mixture wasvigorously shaken for 15 seconds and then left at rest at roomtemperature for 3 minutes. Then, the resulting mixture was centrifugedat 12,000×g at 4° C. for 15 minutes, and each 5 ml of aqueous layer weretransferred to two new tubes. After 5 ml of ISOGEN was added to eachtube, the mixture was immediately shaken vigorously for 15 seconds, andleft at rest at room temperature for 3 minutes. Then, the resultingmixture was centrifuged at 12,000×g at 4° C. for 15 minutes, and each 10ml of aqueous layer were transferred to new 50 ml tubes, respectively.Subsequently, 10 ml of isopropanol (manufactured by Wako Pure ChemicalIndustries, Ltd.) was added to each tube, and the mixture was kept onice for 30 minutes. The resulting mixture was centrifuged at 12,000×g at4° C. for 10 minutes to precipitate RNA. After the supernatant wasremoved, 20 ml of 70% ethanol was added to the residue. The resultingmixture was centrifuged at 10,000×g at 4° C. for 5 minutes. After thesupernatant was removed, the precipitate of total RNA was slightly driedand then dissolved in 1 ml of commercially available RNase-free water(Nacalai Tesque, Inc.). A concentration of the prepared total RNA(calculated from an absorbance at 260 nm) was 6.9 mg/ml.

(2) Extraction of Total RNA from German Cockroach

Adults, nymphs and oothecae of artificially-reared German cockroach(Blattella germanica) were provided as samples. Ten (10) of adult malesand 10 of adult females (individuals from each of which ootheca has beenremoved) were used as an adult sample of 1.1 g, 10 of nymph males and 10of nymph females were used as a nymph sample of 1.0 g, and 26 oothecaewere used as an ootheca sample of 1.0 g. Three kinds of these sampleswere separately crushed into a powder in liquid nitrogen using separatemortars and pestles. From each of the resulting frozen crushed powders,RNA was isolated using a RNA extracting reagent ISOGEN (manufactured byNippon Gene) as follows. After 10 ml of ISOGEN was added to the frozencrushed powder in the mortar, the crushed powder was ground for 10minutes while kept on ice. After grinding, a fluid sample wastransferred to a 15 ml tube with a pipette, and 2 ml of chloroform(manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto.Immediately, the mixture was vigorously shaken for 15 seconds and thenleft at rest at room temperature for 3 minutes. Then, the resultingmixture was centrifuged at 12,000×g at 4° C. for 15 minutes, and each 5ml of aqueous layer were transferred to two new tubes. After 5 ml ofISOGEN was added to each tube, the mixture was immediately shakenvigorously for 15 seconds, and left at rest at room temperature for 3minutes. Then, the resulting mixture was centrifuged at 12,000×g at 4°C. for 15 minutes, and each 10 ml of aqueous layer were transferred tonew 50 ml tubes, respectively. Subsequently, 10 ml of isopropanol(manufactured by Wako Pure Chemical Industries, Ltd.) was added to eachtube, and the mixture was kept on ice for 30 minutes. The resultingmixture was centrifuged at 12,000×g at 4° C. for 10 minutes toprecipitate RNA. After the supernatant was removed, 20 ml of 70% ethanolwas added to the residue. The resulting mixture was centrifuged at10,000×g at 4° C. for 5 minutes. After the supernatant was removed, theprecipitate of total RNA was slightly dried and then dissolved in 1 mlof commercially available RNase-free water (Nacalai Tesque, Inc.). Aconcentration of the prepared total RNA (calculated from absorbance at260 nm) was 1.1 mg/ml in the case of adult-derived total RNA, was 2.5mg/ml in the case of nymph-derived total RNA, and 1.4 mg/ml in the caseof ootheca-derived total RNA.

Example 2 Isolation of Cotton Aphid Choline Acetyltransferase Gene

First-strand cDNA was prepared using total RNA from cotton aphid, randomPrimers (Invitrogen) and Superscript III (Invitrogen) for RT-PCRaccording to the manufacturer's procedure of Superscript III.

A full-length cDNA of cotton aphid choline acetyltransferase wasamplified by PCR using an oligonucleotide comprising the nucleotidesequence of SEQ ID NO: 4 and an oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 5, which are primers specific for thegene, and Pfu Ultra HF Taq polymerase (manufactured by Stratagene)according to the manufacturer's procedure. First-strand cDNA, preparedas described above, was used as template. The PCR conditions are asfollows: an initial denaturation at 94° C. for 5 minutes; followed by 35cycles of PCR, one cycle being 94° C. for 20 seconds, 53° C. for 20seconds, and 72° C. for 3 minutes; followed by 72° C. for 10 minutes.The resulting PCR products were analyzed and purified by agarose gelelectrophoresis to obtain DNA of interest. Further, the obtained DNA wascloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced to determine the nucleotide sequence of 2360 bp shown in SEQID NO: 3. Based on the sequence, an ORF encoding a cotton aphid cholineacetyltransferase was identified as having the nucleotide sequence of2211 bp shown in SEQ ID NO: 2. An amino acid sequence presumed from thenucleotide sequence of SEQ ID NO: 3 was the amino acid sequence of SEQID NO: 1.

Example 3 Isolation of German Cockroach Choline Acetyltransferase Gene

For isolation of homologues of choline acetyltransferase gene from otherinsect species such as German cockroach (Blatella germanica), degenerateprimers are designed using Codehop program (publicly accessible on thewebsite of Blocks Protein Analysis Server operated within the FredHutchinson Cancer Research Center atblocks.fhcrc.org/blocks/codehop.html), and based on the amino acidsequence of the aforementioned cotton aphid choline acetyltransferaseand the previously-known amino acid sequences from such as D.melanogaster (NCBI accession number P07668), Ceanorhabditis elegans(P32756) and Anopheles gambiae (XP_(—)312586).

Partial sequences of a homologue of choline acetyltransferase gene of aselected insect species are amplified by a series of PCR usingfirst-strand cDNA derived from the insect species as a template. Herein,the first-strand cDNA as a template is prepared by the aforementionedmethod using Superscript III. Amplification by PCR is performed using aset of degenerate primers as a forward primer and a reverse primer aswell as Amplitaq Gold (manufactured by Applied Biosystems) according tothe manufacturer's procedure annexed to the reagent. The PCR conditionsare those for touchdown PCR as follows: an initial denaturation at 94°C. for 10 minutes; followed by 10 cycles of touchdown-PCR, one cyclebeing 94° C. for 30 seconds, 60° C. for 1 minute with a decrease of 1°C. per cycle, and 72° C. for 1 minute and 30 seconds; followed by 25cycles of PCR, one cycle being 94° C. for 30 seconds, 50° C. for 1minute, and 72° C. for 1 minute and 30 seconds; and followed by 72° C.for 7 minutes. The PCR product is analyzed and purified by agarose gelelectrophoresis to obtain DNA of interest. Further, the obtained DNA iscloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

Thus, partial sequence of a choline acetyltransferase gene of Blatellagermanica is obtained.

Then, primers specific for the resulting partial sequences of the insecthomologue of choline acetyltransferase gene are designed, and 3′RACE PCRor 5′RACE PCR is performed in order to obtain a full-length sequence ofthe gene. The 3′RACE PCR is performed employing first-strand cDNAprepared from the insect total RNA as a template and using SMART PCRcDNA Synthesis Kit (manufactured by Clontech) according to themanufacturer's instructions annexed to the kit. The 5′RACE PCR isperformed employing first-strand cDNA prepared from the insect total RNAas a template and using 5′/3′ RACE Kit, 2nd Generation (manufactured byRoche) according to the manufacturer's instructions annexed to the kit.

In 3′RACE reaction, a forward primer specific for the sequence ofinterest is used in combination with universal primer mix (UPM)contained in SMART PCR cDNA Synthesis Kit as a reverse primer. The PCRconditions are those for touchdown PCR as follows: an initialdenaturation at 94° C. for 10 minutes; followed by 10 cycles oftouchdown-PCR, one cycle being 94° C. for 20 seconds, 60° C. for 20seconds with a decrease of 1° C. per cycle, and 72° C. for 2 minutes;followed by 25 cycles of PCR, one cycle being 94° C. for 20 seconds, 50°C. for 20 seconds, and 72° C. for 3 minutes; and followed by 72° C. for7 minutes. The resulting PCR product is analyzed and purified by agarosegel electrophoresis to obtain DNA of interest. Further, the obtained DNAis cloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

When a distinct amplification product is not obtained by the first-roundPCR, nested PCR is performed using the first-round PCR product as atemplate. As primers, a specific forward primer which is designed tobind to internal sequence of the first-round PCR product is used incombination with NUP primer contained in SMART PCR cDNA Synthesis Kit asa reverse primer. The PCR conditions are as follows: an initialdenaturation at 95° C. for 10 minutes; followed by 35 cycles of PCR, onecycle being 95° C. for 20 seconds, 50° C. for 20 seconds, and 72° C. for3 minutes; followed by 72° C. for 7 minutes. The resulting PCR productis analyzed and purified by agarose gel electrophoresis to obtain DNA ofinterest. Further, the obtained DNA is cloned into the pCR4-TOPO vector(manufactured by Invitrogen), and sequenced.

In 5′RACE reaction, a reverse primer specific for the sequence ofinterest is used in combination with Oligo-d(T)-anchor primer1 containedin 5′/3′RACE Kit, 2nd Generation as a forward primer. The PCR conditionsare those for touchdown PCR as follows: an initial denaturation at 94°C. for 10 minutes; followed by 10 cycles of touchdown-PCR, one cyclebeing 94° C. for 30 seconds, 58° C. for 30 seconds with a decrease of 1°C. per cycle, and 72° C. for 2 minutes; followed by 25 cycles of PCR,one cycle being 94° C. for 30 seconds, 48° C. for 1 minute, and 72° C.for 2 minutes; and followed by 72° C. for 7 minutes. The resulting PCRproduct is analyzed and purified by agarose gel electrophoresis toobtain DNA of interest. Further, the obtained DNA is cloned into thepCR4-TOPO vector (manufactured by Invitrogen), and sequenced.

When a distinct amplification product is not obtained by the first-roundPCR, nested PCR is performed using the first-round PCR product as atemplate. As primers, a specific reverse primer which is designed tobind to internal sequence of the first-round PCR product is used incombination with PCR Anchor primer contained in 5′/3′RACE Kit, 2ndGeneration as a forward primer. The PCR conditions are as follows: aninitial denaturation at 94° C. for 10 minutes; followed by 10 cycles ofPCR, one cycle being 94° C. for 15 seconds, 55° C. for 30 seconds, and72° C. for 40 seconds; followed by 25 cycles of PCR, one cycle being 94°C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 40 secondsextended with 20 seconds each cycle; followed by 72° C. for 7 minutes.The resulting PCR product is analyzed and purified by agarose gelelectrophoresis to obtain DNA of interest. Further, the obtained DNA iscloned into the pCR4-TOPO vector (manufactured by Invitrogen), andsequenced.

The above sequencing results reveal 5′-terminal sequence and 3′-terminalsequence, each encoding N-terminal region and C-terminal region of theinsect choline acetyltransferase, respectively.

Example 4 Construction of Recombinant Plasmid

A choline acetyltransferase gene fragment to be cloned into a vector forexpression in E. coli was amplified by PCR using an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 4 and anoligonucleotide comprising the nucleotide sequence of SEQ ID NO: 5,which are primers specific for the gene, and Pfu Ultra HF Taq polymerase(manufactured by Stratagene) according to the manufacturer's procedure.The cDNA obtained in Example 2 was used as template. The PCR conditionsused were as follows: an initial denaturation at 94° C. for 5 minutes;followed by 35 cycles of PCR, one cycle being 94° C. for 20 seconds, 53°C. for 20 seconds, and 72° C. for 3 minutes; followed by 72° C. for 10minutes.

The resulting PCR product was purified using the Qiaquick PCRPurification Kit (manufactured by Qiagen) in accordance with theinstruction attached to the kit. The DNA fragment after the purificationwas digested with Bam HI and XhoI, since the oligonucleotide of SEQ IDNO: 4 contains a Bam HI restriction site and the oligonucleotide of SEQID NO: 5 contains a Xho I restriction site.

The Bam HI/XhoI DNA fragment of cotton aphid choline acetyltransferasegene was analyzed by an agarose gel electrophoresis, isolated, purifiedand ligated into the Bam HI/XhoI cloning sites of the E. coli expressionvector pET41a(+) (manufactured by Novagen). The obtained vector wascalled pGBJ005.

Translation of the recombinant choline acetyltransferase together withtwo His-tags and one GST-tag provided a recombinant protein of 1021amino acids.

Following the procedures described in Qiagen Plasmid PurificationHandbook, pGBJ005 was prepared using a Qiafilter Plasmid Maxiprep(Qiagen).

Example 5 Preparation of Recombinant E. coli

Competent cells of E. coli BL21 (DE3) (manufactured by Invitrogen) weretransformed following the manufacturers instruction, using 1 μl ofpGBJ005 at a concentration of 1 ng/μl. Colonies of the transformed E.coli were grown on LB agar plates containing 50 mg/L Kanamycin(manufactured by Sigma) at 37° C. overnight.

Example 6 Expression of Choline Acetyltransferase in E. Coli

E. coli BL21 (DE3) transformed with pGBJ005 was rotary culturedovernight in LB-medium containing 50 mg/L of Kanamycin (manufactured bySigma) at 37° C., 250 rpm. In the next morning, the culture was diluted1/100 in LB medium containing 50 mg/L of Kanamycin, to which IPTG wasadded until a final concentration of 10 μM, and grown at 22° C., 60 rpmfor 4 days to produce recombinant choline acetyltransferase protein inthe E. coli. The culture was centrifuged at 7,000 rpm for 10 minutes tocollect the E. coli. The supernatant was discarded, and the remaining E.coli was flash frozen in liquid nitrogen and stored at −80° C. untilusage.

Example 7 Purification of Choline Acetyltransferase

The cotton aphid choline acetyltransferase was cloned in pET41a(+) inframe with a N-terminal GST (glutathione S-transferase)-tag and His-tagand a C-terminal 6×His-tag (SEQ ID NO: 6). The recombinant cholineacetyltransferase protein was purified utilizing His-tag.

(1) Preparation of Crude Extract

The frozen cell pellets of induced E. coli BL21(DE3) cell wereresuspended in 30 ml of breaking buffer (0.1M Sodium phosphate buffer pH7.6, 1 tablet of Complete EDTA-free protease inhibitor cocktail(manufactured by Roch)), and subsequently lysed in breaking buffer byusing French press (manufactured by Thermo Spectronic). The pressure wasmaintained at 1300 to 1500 psi during the procedure of breaking of thecells. The French pressed solution was centrifuged for 60 minutes at14,000 rpm at 2° C. to collect a supernatant. The collected supernatantwas filtered through a 0.45 μm filter and kept on ice.

(2) Purification Utilizing His-Tag

The recombinant protein was purified by utilizing metal affinitychromatography, using either the HiTrap Chelating HP (Amershambiosciences) or HisTrap HP (Amersham biosciences) columns, according tothe instructions of the manufacturer (Amersham biosciences). For largerscale purifications, a XK-16/20 column (Amersham biosciences) was used,the column being filled with Chelating Fast Flow Sepharose (Amershambiosciences). The purification procedure was undertaken on the AKTA-FPLC(Amersham biosciences).

Hitrap, HisTrap, and AX-16/20 affinity columns have been preparedaccording to the manufacturer's protocol. Buffer A, the binding buffer,was made of 0.1M Sodium phosphate buffer pH 7.6, and 10% glycerol.Buffer B, the elution buffer, was made of 0.1M Sodium phosphate bufferpH 7.6, 500 mM imidazole, and 10% glycerol.

The purification of cotton aphid choline acetyltransferase utilizing aHis-tag was performed as the following procedure:

(i) sample injection;(ii) washing out unbound sample with 5 column volumes (CV) of 95% bufferA/5% buffer B (25 mM imidazole);(iii) washing for 15 CV of 90% buffer A/10% buffer B (50 mM imidazole);(iv) washing for 15 CV of 85% buffer A/15% buffer B (75 mM imidazole);(v) washing for 15 CV of 80% buffer A/20% buffer B (100 mM imidazole);(vi) elusion of purified protein with 15 CV of 60% buffer A/40% buffer B(200 mM imidazole); and(vii) washing the column with 5 CV of 100% buffer B (500 mM imidazole).

The fractions obtained from the elution with 60% buffer A/40% buffer Bwere pooled and stored on ice.

The obtained elution fractions were analysed to verify presence of therecombinant cotton aphid choline acetyltransferase protein. An 8%polyacrylamide gel was used for optimal gel electrophoresis resolutionof the expressed choline acetyltransferase protein of 115 kDa.

The following staining solution and destain solution were used forCoomassie staining of polyacrylamide gel. Staining solution was made of1 g/l Coomassie Brilliant blue R, 50 (v/v) % Methanol, 12 (v/v) % Aceticacid and 38 (v/v) % distilled water. After mixing, the solution wasfiltered to get out unsoluble Brilliant blue R dye. Destain solution wasmade of 25 (v/v) % Methanol, 10 (v/v) % Acetic acid and 65 (v/v) %distilled water.

For western blot analysis, an anti-His (H15) sc-803 rabbit polyclonalIgG antibody (tebubio) was used as primary antibody at a 1:500 dilution.The secondary antibody was a goat anti-rabbit-HRP (Pierce) at a dilutionof 1:10000.

After analysis of the polyacrylamide gels by SDS-PAGE and Westernblotting, the fractions of interest were pooled and the proteinconcentration was determined. Protein Concentration was determined byBradford method with the Bradford Bio-Rad protein assay (Bio-Rad) usingPre-diluted Protein Assay Standards (Pierce): Bovine Serum AlbuminFraction V Set according to the manufacturer's protocol. The pooledfractions were then distributed into several aliquots and immediatelyflash-frozen in liquid nitrogen and stored at −80° C.

Example 8 Selection of Compounds Inhibiting Choline AcetyltransferaseActivity

Selection of a compound which modulates a choline acetyltransferaseactivity was performed in a system for measuring and evaluating thecholine acetyltransferase activity, the activity being modulated byadding a test compound to an in vitro reaction system using the cottonaphid choline acetyltransferase prepared in Example 7.

As for a measurement of the cotton aphid choline acetyltransferaseactivity, after the enzymatic reaction of choline acetyltransferaseusing acetyl-CoA and choline as substrates, free CoA formed by cholineacetyltransferase reaction was measured using5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) reagent. DTNB reacts withfree thiol groups in solution to produce 5-thio-2-nitrobenzoic acid(TNB). TNB is yellow and has absorption maximum at 412 nm. The producedTNB was measured calorimetrically to calculate choline acetyltransferaseactivity.

For measuring the activity, the activity of the aphid cholineacetyltransferase was measured when a test compound dissolved in DMSOwas contained to a final concentration of 10 μM. In addition, theactivity of the aphid choline acetyltransferase was measured when DMSOwas contained in place of a test compound. Then, a ratio (%) of ameasured value of the activity of the aphid choline acetyltransferasewhen a test compound dissolved in DMSO was contained, relative to ameasured value of the activity of aphid choline acetyltransferase whenDMSO was contained in place of the test compound was calculated, and avalue obtained by subtracting the calculated value from 100% was adoptedas an inhibition degree (%). The results in each test compound are shownin Table 4 in Example 9 together with results of Example 9.

The activity of aphid choline acetyltransferase was measured when a testcompound dissolved in DMSO was contained to a final concentration ofeach concentration of 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μMor 0.03 μM. IC₅₀ (μM) was calculated from the result of eachconcentration at each test compound using a concentration-dependent testanalyzing software XL fit (manufactured by idbs). The results are shownin Table 5 in Example 10 together with results of Example 9.

Example 9 Pesticidal Activity Test

A sterilized artificial feed having the following composition (Table 3)was prepared. Then, according to the same manner as that of the methoddescribed in Handbook of Insect Rearing Vol. 1 (Elsevier SciencePublishers 1985) pp. 35 to pp. 36 except that a test compound dissolvedin DMSO to a final concentration of 50 ppm was added at 0.5% volume ofthe artificial feed, and components were mixed, Aphis gossypii wasreared. Six days after rearing, the number of surviving Aphis gossypiiwas investigated, and an entity exhibiting a significant controllingvalue (e.g. controlling value of 30% or more) was determined to havepesticidal activity by obtaining a controlling value by the followingequation.

Controlling value(%)={1−(Cb×Tai)/(Cai×Tb)}×100

Letters in the equation represent the following meanings.

Cb: Number of surviving worms before treatment in non-treated sectionCai: Number of surviving worms at observation in non-treated sectionTb: Number of surviving worms before treatment in treated sectionTai: Number of surviving worms at observation in treated section

Results are shown in Table 4 in Example 9 together with results ofExample 8.

TABLE 3 Amino acid (mg/100 ml) L-alanine 100.0 L-arginine 275.0L-asparagine 550.0 L-aspartic acid 140.0 L-cysteine 40.0 (hydrochloride)L-glutamic acid 140.0 L-glutamine 150.0 L-glycine 80.0 L-histidine 80.0L-isoleucine 80.0 L-leucine 80.0 L-lysine 120.0 (hydrochloride)L-methionine 80.0 L-phenylalanine 40.0 L-proline 80.0 L-serine 80.0L-threonine 140.0 L-tryptophan 80.0 L-tyrosine 40.0 L-valine 80.0Vitamins (mg/100 ml) Ascorbic acid 100.0 Biotin 0.1 Calcium 5.0pantothenate Choline chloride 50.0 Inositol 50.0 Nicotinic acid 10.0Thiamine 2.5 Others (mg/100 ml) Sucrose 12500.0 Dipotassium 1500.0hydrogen phosphate Magnesium sulfate 123.0 Cupric chloride 0.2 Ferricchloride 11.0 Manganese chloride 0.4 Zinc sulfate 0.8 (anhydrous)Adjusted to pH 6.8

TABLE 4 DD03493

DD05314

Result of Example 8 Activity of inhibiting choline acetyltransferaseactivity Result of Example 9 (inhibition degree (%) Determination resultCompound at 10 μM addition) of pesticidal activity DD03493 49 Presenceof pesticidal activity DD05314 50 Presence of pesticidal activityDF07204 71 Presence of pesticidal activity

Example 10 Pesticidal Activity Test

According to the same manner as that of Example 9, pesticidal activitytest was performed, and results are shown in Table 5 in Example 10together with results of Example 8.

TABLE 5 Result of Example 8 Activity of Result of Example 10 inhibitingcholine Determination acetyltransferase result of pesticidal Compoundactivity (IC50, μM) activity 2-(α-naphthoyl)ethyl- 33.5 Presence ofdimethyl ammonium pesticidal activity chloride (α-NEDA)2-(α-naphthoyl)ethyl- 1.5 Presence of trimethylammonium pesticidalactivity iodide(α-NETA) DD03493 23.8 Presence of pesticidal activityCisapride >100 Absence of pesticidal activity DL-Chlorpheniramine >100Absence of Maleate pesticidal activity

INDUSTRIAL APPLICABILITY

According to the present invention, it becomes possible to provide amore target-based approach of screening agricultural chemicals, wherebycompounds are screened against a specific target with intent ofchemically interfering with the target site to control insects or otherpest organisms.

Free Text in Sequence Listing SEQ ID NO: 4

Designed oligonucleotide primer for PCR

SEQ ID NO: 5

Designed oligonucleotide primer for PCR

1. An isolated insect choline acetyltransferase comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO: 1; (b) an amino acid sequence with deletion,addition or substitution of one or more amino acids in the amino acidsequence of SEQ ID NO: 1, wherein said amino acid sequence has cholineacetyltransferase activity; (c) an amino acid sequence that has sequenceidentity of 50% or more to the amino acid sequence of SEQ ID NO: 1,wherein said amino acid sequence has choline acetyltransferase activity;(d) an amino acid sequence that has sequence similarity of 75% or moreto the amino acid sequence of SEQ ID NO: 1, wherein said amino acidsequence has choline acetyltransferase activity; (e) the amino acidsequence encoded by the nucleotide sequence of SEQ ID NO: 2 or 3; (f) anamino acid sequence encoded by a nucleotide sequence that has sequenceidentity of 50% or more to the nucleotide sequence of SEQ ID NO: 2 or 3,wherein said amino acid sequence has choline acetyltransferase activity;(g) an amino acid sequence encoded by a polynucleotide, wherein saidpolynucleotide hybridizes under a stringent condition to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 2 or 3, wherein said amino acidsequence has choline acetyltransferase activity; and (h) an amino acidsequence of a cotton aphid choline acetyltransferase.
 2. An isolatedinsect choline acetyltransferase comprising an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence ofSEQ ID NO: 1; (b) an amino acid sequence that has sequence identity ofat least 90% to the amino acid sequence of SEQ ID NO: 1, wherein saidamino acid sequence has choline acetyltransferase activity; (c) an aminoacid sequence that has sequence similarity of at least 90% to the aminoacid sequence of SEQ ID NO: 1, wherein said amino acid sequence hascholine acetyltransferase activity; (d) the amino acid sequence encodedby the nucleotide sequence of SEQ ID NO: 2 or 3; (e) an amino acidsequence encoded by a nucleotide sequence that has sequence identity ofat least 90% to the nucleotide sequence of SEQ ID NO: 2 or 3, whereinsaid amino acid sequence has choline acetyltransferase activity; (f) anamino acid sequence encoded by a polynucleotide, wherein saidpolynucleotide hybridizes under stringency conditions comprising a saltconcentration of about 6.0×SSC and a temperature of about 65° C. to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 2 or 3, wherein said amino acidsequence has choline acetyltransferase activity; and (g) an amino acidsequence of a cotton aphid choline acetyltransferase.
 3. The cholineacetyltransferase of claim 2, which is a protein comprising the aminoacid sequence of SEQ ID NO:
 1. 4. The choline acetyltransferase of claim2, which is a protein comprising an amino acid sequence that hassequence identity of at least 90% to the amino acid sequence of SEQ IDNO: 1, wherein said protein has choline acetyltransferase activity. 5.The choline acetyltransferase of claim 2, which is a protein comprisingan amino acid sequence that has sequence similarity of at least 90% tothe amino acid sequence of SEQ ID NO: 1, wherein said protein hascholine acetyltransferase activity.
 6. The choline acetyltransferase ofclaim 2, which is a protein comprising the amino acid sequence encodedby the nucleotide sequence of SEQ ID NO: 2 or
 3. 7. The cholineacetyltransferase of claim 2, which is a protein comprising an aminoacid sequence encoded by a nucleotide sequence that has sequenceidentity of at least 90% to the nucleotide sequence of SEQ ID NO: 2 or3, wherein said protein has choline acetyltransferase activity.
 8. Thecholine acetyltransferase of claim 2, which is a cotton aphid cholineacetyltransferase.